Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

ABSTRACT

Methods for preparing cell lines that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, and methods for delivery of the chromosomes to selected cells and tissues are provided. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods. Methods for use of the artificial chromosomes are also provided.

RELATED APPLICATIONS

This application is a divisional of copending U.S. application Ser. No.09/724,872, filed Nov. 28, 2000, to GYULA HADLACZKY and ALADAR SZALAY,entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARINGARTIFICIAL CHROMOSOMES.

This application is a continuation of copending U.S. application Ser.No. 08/835,682, now abandoned, filed Apr. 10, 1997, to GYULA HADLACZKYand ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF ANDMETHODS FOR PREPARING ARTIFICIAL CHROMOSOMES. This application is also acontinuation-in-part of U.S. application Ser. No. 08/695,191, filed Aug.7, 1996, now U.S. Pat. No. 6,025,155, to GYULA HADLACZKY and ALADARSZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FORPREPARING ARTIFICIAL CHROMOSOMES. This application is alsocontinuation-in-part of U.S. application Ser. No. 08/682,080, filed Jul.15, 1996, now U.S. Pat. No. 6,077,697, to GYULA HADLACZKY and ALADARSZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FORPREPARING ARTIFICIAL CHROMOSOMES, and is also a continuation-in-part ofcopending U.S. application Ser. No. 08/629,822, now abandoned, filedApr. 10, 1996 to GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIALCHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIALCHROMOSOMES.

U.S. application Ser. No. 08/835,682 is a continuation-in-part of U.S.application Ser. No. 08/695,191. U.S. application Ser. No. 08/695,191 isa continuation-in-part of U.S. application Ser. No. 08/682,080 and alsois a continuation-in-part of U.S. application Ser. No. 08/629,822. U.S.application Ser. No. 08/682,080 is a continuation-in-part of U.S.application Ser. No. 08/629,822.

This application is related to U.S. application Ser. No. 07/759,558, nowU.S. Pat. No. 5,288,625, is related to U.S. application Ser. No.08/734,344, filed Oct. 21, 1996, and is related to U.S. application Ser.No. 08/375,271, filed Jan. 19, 1995, now U.S. Pat. No. 5,712,134. U.S.application Ser. No. 08/375,271 is a continuation of U.S. applicationSer. No. 08/080,097, filed Jun. 23, 1993 which is a continuation of U.S.application Ser. No. 07/892,487, filed Jun. 3, 1992, which is acontinuation of U.S. application Ser. No. 07/521,073, filed May 9, 1990.

The subject matter of each of the above-noted U.S. applications andpatents is incorporated in its entirety by reference thereto.

The subject matter of each of the above-noted U.S. applications andpatents is incorporated in its entirety by reference thereto.

FIELD OF THE INVENTION

The present invention relates to methods for preparing cell lines thatcontain artificial chromosomes, methods for isolation of the artificialchromosomes, targeted insertion of heterologous DNA into thechromosomes, delivery of the chromosomes to selected cells and tissuesand methods for isolation and large-scale production of the chromosomes.Also provided are cell lines for use in the methods, and cell lines andchromosomes produced by the methods. Further provided are cell-basedmethods for production of heterologous proteins, gene therapy methodsand methods of generating transgenic animals, particularly non-humantransgenic animals, that use artificial chromosomes.

BACKGROUND OF THE INVENTION

Several viral vectors, non-viral, and physical delivery systems for genetherapy and recombinant expression of heterologous nucleic acids havebeen developed [see, e.g., Mitani et al. (1993) Trends Biotech.11:162-166]. The presently available systems, however, have numerouslimitations, particularly where persistent, stable, or controlled geneexpression is required. These limitations include: (1) size limitationsbecause there is a limit, generally on order of about ten kilobases[kB], at most, to the size of the DNA insert [gene] that can be acceptedby viral vectors, whereas a number of mammalian genes of possibletherapeutic importance are well above this limit, especially if allcontrol elements are included; (2) the inability to specifically targetintegration so that random integration occurs which carries a risk ofdisrupting vital genes or cancer suppressor genes; (3) the expression ofrandomly integrated therapeutic genes may be affected by the functionalcompartmentalization in the nucleus and are affected by chromatin-basedposition effects; (4) the copy number and consequently the expression ofa given gene to be integrated into the genome cannot be controlled.Thus, improvements in gene delivery and stable expression systems areneeded [see, e.g., Mulligan (1993) Science 260:926-932].

In addition, safe and effective vectors and gene therapy methods shouldhave numerous features that are not assured by the presently availablesystems. For example, a safe vector should not contain DNA elements thatcan promote unwanted changes by recombination or mutation in the hostgenetic material, should not have the potential to initiate deleteriouseffects in cells, tissues, or organisms carrying the vector, and shouldnot interfere with genomic functions. In addition, it would beadvantageous for the vector to be non-integrative, or designed forsite-specific integration. Also, the copy number of therapeutic gene(s)carried by the vector should be controlled and stable, the vector shouldsecure the independent and controlled function of the introducedgene(s); and the vector should accept large (up to Mb size) inserts andensure the functional stability of the insert.

The limitations of existing gene delivery technologies, however, arguefor the development of alternative vector systems suitable fortransferring large [up to Mb size or larger] genes and gene complexestogether with regulatory elements that will provide a safe, controlled,and persistent expression of the therapeutic genetic material.

At the present time, none of the available vectors fulfill all theserequirements. Most of these characteristics, however, are possessed bychromosomes. Thus, an artificial chromosome would be an ideal vector forgene therapy, as well as for stable, high-level, controlled productionof gene products that require coordination of expression of numerousgenes or that are encoded by large genes, and other uses. Artificialchromosomes for expression of heterologous genes in yeast are available,but construction of defined mammalian artificial chromosomes has notbeen achieved. Such construction has been hindered by the lack of anisolated, functional, mammalian centromere and uncertainty regarding therequisites for its production and stable replication. Unlike in yeast,there are no selectable genes in close proximity to a mammaliancentromere, and the presence of long runs of highly repetitivepericentric heterochromatic DNA makes the isolation of a mammaliancentromere using presently available methods, such as chromosomewalking, virtually impossible. Other strategies are required forproduction of mammalian artificial chromosomes, and some have beendeveloped. For example, U.S. Pat. No. 5,288,625 provides a cell linethat contains an artificial chromosome, a minichromosome, that is about20 to 30 megabases. Methods provided for isolation of these chromosomes,however, provide preparations of only about 10-20% purity. Thus,development of alternative artificial chromosomes and perfection ofisolation and purification methods as well as development of moreversatile chromosomes and further characterization of theminichromosomes is required to realize the potential of this technology.

Therefore, it is an object herein to provide mammalian artificialchromosomes and methods for introduction of foreign DNA into suchchromosomes. It is also an object herein to provide methods of isolationand purification of the chromosomes. It is also an object herein toprovide methods for introduction of the mammalian artificial chromosomeinto selected cells, and to provide the resulting cells, as well astransgenic non-human animals, birds, fish and plants that contain theartificial chromosomes. It is also an object herein to provide methodsfor gene therapy and expression of gene products using artificialchromosomes. It is a further object herein to provide methods forconstructing species-specific artificial chromosomes de novo. Anotherobject herein is to provide methods to generate de novo mammalianartificial chromosomes.

SUMMARY OF THE INVENTION

Mammalian artificial chromosomes [MACs] are provided. Also provided areartificial chromosomes for other higher eukaryotic species, such asinsects, birds, fowl and fish, produced using the MACS and methodsprovided herein. Methods for generating and isolating such chromosomesare provided. Methods using the MACs to construct artificial chromosomesfrom other species, such as insect, bird, fowl and fish species are alsoprovided. The artificial chromosomes are fully functional stablechromosomes. Two types of artificial chromosomes are provided. One type,herein referred to as SATACs [satellite artificial chromosomes orsatellite DNA based artificial chromosomes (the terms are usedinterchangeably herein)] are stable heterochromatic chromosomes, and theother type are minichromosomes based on amplification of euchromatin.

Artificial chromosomes provide an extra-genomic locus for targetedintegration of megabase [Mb] pair size DNA fragments that contain singleor multiple genes, including multiple copies of a single geneoperatively linked to one promoter or each copy or several copies linkedto separate promoters. Thus, methods using the MACs to introduce thegenes into cells, tissues, and animals, as well as species such asbirds, fowl, fish and plants, are also provided. The artificialchromosomes with integrated heterologous DNA may be used in methods ofgene therapy, in methods of production of gene products, particularlyproducts that require expression of multigenic biosynthetic pathways,and also are intended for delivery into the nuclei of germline cells,such as embryo-derived stem cells [ES cells], for production oftransgenic (non-human) animals, birds, fowl and fish. Transgenic plants,including monocots and dicots, are also contemplated herein.

Mammalian artificial chromosomes provide extra-genomic specificintegration sites for introduction of genes encoding proteins ofinterest and permit megabase size DNA integration so that, for example,genes encoding an entire metabolic pathway or a very large gene, such asthe cystic fibrosis [CF; ˜250 kb] genomic DNA gene, several genes, suchas multiple genes encoding a series of antigens for preparation of amultivalent vaccine, can be stably introduced into a cell. Vectors fortargeted introduction of such genes, including the tumor suppressorgenes, such as p53, the cystic fibrosis transmembrane regulator cDNA[CFTR], and the genes for anti-HIV ribozymes, such as an anti-HIV gagribozyme gene, into the artificial chromosomes are also provided.

The chromosomes provided herein are generated by introducingheterologous DNA that includes DNA encoding one or multiple selectablemarker(s) into cells, preferably a stable cell line, growing the cellsunder selective conditions, and identifying from among the resultingclones those that include chromosomes with more than one centromereand/or fragments thereof. The amplification that produces the additionalcentromere or centromeres occurs in cells that contain chromosomes inwhich the heterologous DNA has integrated near the centromere in thepericentric region of the chromosome. The selected clonal cells are thenused to generate artificial chromosomes.

Although non-targeted introduction of DNA, which results in somefrequency of integration into appropriate loci, targeted introduction ispreferred. Hence, in preferred embodiments, the DNA with the selectablemarker that is introduced into cells to initiate generation ofartificial chromosomes includes sequences that target it to the anamplifiable region, such as the pericentric region, heterochromatin, andparticularly rDNA of the chromosome. For example, vectors, such aspTEMPUD and pHASPUD [provided herein], which include such DNA specificfor mouse satellite DNA and human satellite DNA, respectively, areprovided. The plasmid pHASPUD is a derivative of pTEMPUD that containshuman satellite DNA sequences that specifically target humanchromosomes. Preferred targeting sequences include mammalian ribosomalRNA (rRNA) gene sequences (referred to herein as rDNA) which target theheterologous DNA to integrate into the rDNA region of those chromosomesthat contain rDNA. For example, vectors, such as pTERPUD, which includemouse rDNA, are provided. Upon integration into existing chromosomes inthe cells, these vectors can induce the amplification that results ingeneration of additional centromeres.

Artificial chromosomes are generated by culturing the cells with themulticentric, typically dicentric, chromosomes under conditions wherebythe chromosome breaks to form a minichromosome and formerly dicentricchromosome. Among the MACs provided herein are the SATACs, which areprimarily made up of repeating units of short satellite DNA and arenearly fully heterochromatic, so that without insertion of heterologousor foreign DNA, the chromosomes preferably contain no geneticinformation or contain only non-protein-encoding gene sequences such asrDNA sequences. They can thus be used as “safe” vectors for delivery ofDNA to mammalian hosts because they do not contain any potentiallyharmful genes. The SATACs are generated, not from the minichromosomefragment as, for example, in U.S. Pat. No. 5,288,625, but from thefragment of the formerly dicentric chromosome.

In addition, methods for generating euchromatic minichromosomes and theuse thereof are also provided herein. Methods for generating one type ofMAC, the minichromosome, previously described in U.S. Pat. No.5,288,625, and the use thereof for expression of heterologous DNA areprovided. In a particular method provided herein for generating a MAC,such as a minichromosome, heterologous DNA that includes mammalian rDNAand one or more selectable marker genes is introduced into cells whichare then grown under selective conditions. Resulting cells that containchromosomes with more than one centromere are selected and culturedunder conditions whereby the chromosome breaks to form a minichromosomeand a formerly multicentric (typically dicentric) chromosome from whichthe minichromosome was released.

Cell lines containing the minichromosome and the use thereof for cellfusion are also provided. In one embodiment, a cell line containing themammalian minichromosome is used as recipient cells for donor DNAencoding a selected gene or multiple genes. To facilitate integration ofthe donor DNA into the minichromosome, the recipient cell linepreferably contains the minichromosome but does not also contain theformerly dicentric chromosome. This may be accomplished by methodsdisclosed herein such as cell fusion and selection of cells that containa minichromosome and no formerly dicentric chromosome. The donor DNA islinked to a second selectable marker and is targeted to and integratedinto the minichromosome. The resulting chromosome is transferred by cellfusion into an appropriate recipient cell line, such as a Chinesehamster cell line [CHO]. After large-scale production of the cellscarrying the engineered chromosome, the chromosome is isolated. Inparticular, metaphase chromosomes are obtained, such as by addition ofcolchicine, and they are purified from the cell lysate. Thesechromosomes are used for cloning, sequencing and for delivery ofheterologous DNA into cells.

Also provided are SATACs of various sizes that are formed by repeatedculturing under selective conditions and subcloning of cells thatcontain chromosomes produced from the formerly dicentric chromosomes.The exemplified SATACs are based on repeating DNA units that are about15 Mb [two ˜7.5 Mb blocks]. The repeating DNA unit of SATACs formed fromother species and other chromosomes may vary, but typically would be onthe order of about 7 to about 20 Mb. The repeating DNA units arereferred to herein as megareplicons, which in the exemplified SATACscontain tandem blocks of satellite DNA flanked by non-satellite DNA,including heterologous DNA and non-satellite DNA. Amplification producesan array of chromosome segments [each called an amplicon] that containtwo inverted megareplicons bordered by heterologous [“foreign”] DNA.Repeated cell fusion, growth on selective medium and/or BrdU[5-bromodeoxyuridine] treatment or other treatment with other genomedestabilizing reagent or agent, such as ionizing radiation, includingX-rays, and subcloning results in cell lines that carry stableheterochromatic or partially heterochromatic chromosomes, including a150-200 Mb “sausage” chromosome, a 500-1000 Mb gigachromosome, a stable250-400 Mb megachromosome and various smaller stable chromosomes derivedtherefrom. These chromosomes are based on these repeating units and caninclude heterologous DNA that is expressed.

Thus, methods for producing MACs of both types (i.e., SATACS andminichromosomes) are provided. These methods are applicable to theproduction of artificial chromosomes containing centromeres derived fromany higher eukaryotic cell, including mammals, birds, fowl, fish,insects and plants.

The resulting chromosomes can be purified by methods provided herein toprovide vectors for introduction of heterologous DNA into selected cellsfor production of the gene product(s) encoded by the heterologous DNA,for production of transgenic (non-human) animals, birds, fowl, fish andplants or for gene therapy.

In addition, methods and vectors for fragmenting the minichromosomes andSATACs are provided. Such methods and vectors can be used for in vivogeneration of smaller stable artificial chromosomes. Vectors forchromosome fragmentation are used to produce an artificial chromosomethat contains a megareplicon, a centromere and two telomeres and will bebetween about 7.5 Mb and about 60 Mb, preferably between about 10 Mb-15Mb and 30-50 Mb. As exemplified herein, the preferred range is betweenabout 7.5 Mb and 50 Mb. Such artificial chromosomes may also be producedby other methods.

Isolation of the 15 Mb [or 30 Mb amplicon containing two 15 Mb invertedrepeats] or a 30 Mb or higher multimer, such as 60 Mb, thereof shouldprovide a stable chromosomal vector that can be manipulated in vitro.Methods for reducing the size of the MACs to generate smaller stableself-replicating artificial chromosomes are also provided.

Also provided herein, are methods for producing mammalian artificialchromosomes, including those provided herein, In vitro, and theresulting chromosomes. The methods involve in vitro assembly of thestructural and functional elements to provide a stable artificialchromosome. Such elements include a centromere, two telomeres, at leastone origin of replication and filler heterochromatin, e.g., satelliteDNA. A selectable marker for subsequent selection is also generallyincluded. These specific DNA elements may be obtained from theartificial chromosomes provided herein such as those that have beengenerated by the introduction of heterologous DNA into cells and thesubsequent amplification that leads to the artificial chromosome,particularly the SATACs. Centromere sequences for use in the in vitroconstruction of artificial chromosomes may also be obtained by employingthe centromere cloning methods provided herein. In preferredembodiments, the sequences providing the origin of replication, inparticular, the megareplicator, are derived from rDNA. These sequencespreferably include the rDNA origin of replication and amplificationpromoting sequences.

Methods and vectors for targeting heterologous DNA into the artificialchromosomes are also provided as are methods and vectors for fragmentingthe chromosomes to produce smaller but stable and self-replicatingartificial chromosomes.

The chromosomes are introduced into cells to produce stable transformedcell lines or cells, depending upon the source of the cells.Introduction is effected by any suitable method including, but notlimited to electroporation, direct uptake, such as by calcium phosphateprecipitation, uptake of isolated chromosomes by lipofection, bymicrocell fusion, by lipid-mediated carrier systems or other suitablemethod. The resulting cells can be used for production of proteins inthe cells. The chromosomes can be isolated and used for gene delivery.Methods for isolation of the chromosomes based on the DNA content of thechromosomes, which differs in MACs versus the authentic chromosomes, areprovided. Also provided are methods that rely on content, particularlydensity, and size of the MACs.

These artificial chromosomes can be used in gene therapy, gene productproduction systems, production of humanized genetically transformedanimal organs, production of transgenic plants and animals (non-human),including mammals, birds, fowl, fish, invertebrates, vertebrates,reptiles and insects, any organism or device that would employchromosomal elements as information storage vehicles, and also foranalysis and study of centromere function, for the production ofartificial chromosome vectors that can be constructed in vitro, and forthe preparation of species-specific artificial chromosomes. Theartificial chromosomes can be introduced into cells usingmicroinjection, cell fusion, microcell fusion, electroporation, nucleartransfer, electrofusion, projectile bombardment, nuclear transfer,calcium phosphate precipitation, lipid-mediated transfer systems andother such methods. Cells particularly suited for use with theartificial chromosomes include, but are not limited to plant cells,particularly tomato, arabidopsis, and others, insect cells, includingsilk worm cells, insect larvae, fish, reptiles, amphibians, arachnids,mammalian cells, avian cells, embryonic stem cells, haematopoietic stemcells, embryos and cells for use in methods of genetic therapy, such aslymphocytes that are used in methods of adoptive immunotherapy and nerveor neural cells. Thus methods of producing gene products and transgenic(non-human) animals and plants are provided. Also provided are theresulting transgenic animals and plants.

Exemplary cell lines that contain these chromosomes are also provided.

Methods for preparing artificial chromosomes for particular species andfor cloning centromeres are also provided. For example, two exemplarymethods provided for generating artificial chromosomes for use indifferent species are as follows. First, the methods herein may beapplied to different species. Second, means for generatingspecies-specific artificial chromosomes and for cloning centromeres areprovided. In particular, a method for cloning a centromere from ananimal or plant is provided by preparing a library of DNA fragments thatcontain the genome of the plant or animal and introducing each of thefragments into a mammalian satellite artificial chromosome [SATAC] thatcontains a centromere from a species, generally a mammal, different fromthe selected plant or animal, generally a non-mammal, and a selectablemarker. The selected plant or animal is one in which the mammalianspecies centromere does not function. Each of the SATACs is introducedinto the cells, which are grown under selective conditions, and cellswith SATACs are identified. Such SATACS should contain a centromereencoded by the DNA from the library or should contain the necessaryelements for stable replication in the selected species.

Also provided are libraries in which the relatively large fragments ofDNA are contained on artificial chromosomes.

Transgenic (non-human) animals, invertebrates and vertebrates, plantsand insects, fish, reptiles, amphibians, arachnids, birds, fowl, andmammals are also provided. Of particular interest are transgenic(non-human) animals and plants that express genes that confer resistanceor reduce susceptibility to disease. For example, the transgene mayencode a protein that is toxic to a pathogen, such as a virus, bacteriumor pest, but that is not toxic to the transgenic host. Furthermore,since multiple genes can be introduced on a MAC, a series of genesencoding an antigen can be introduced, which upon expression will serveto immunize [in a manner similar to a multivalent vaccine] the hostanimal against the diseases for which exposure to the antigens provideimmunity or some protection.

Also of interest are transgenic (non-human) animals that serve as modelsof certain diseases and disorders for use in studying the disease anddeveloping therapeutic treatments and cures thereof. Such animal modelsof disease express genes [typically carrying a disease-associatedmutation], which are introduced into the animal on a MAC and whichinduce the disease or disorder in the animal. Similarly, MACs carryinggenes encoding antisense RNA may be introduced into animal cells togenerate conditional “knock-out” transgenic (non-human) animals. In suchanimals, expression of the antisense RNA results in decreased orcomplete elimination of the products of genes corresponding to theantisense RNA. Of further interest are transgenic mammals that harborMAC-carried genes encoding therapeutic proteins that are expressed inthe animal's milk. Transgenic (non-human) animals for use inxenotransplantation, which express MAC-carried genes that serve tohumanize the animal's organs, are also of interest. Genes that might beused in humanizing animal organs include those encoding human surfaceantigens.

Methods for cloning centromeres, such as mammalian centromeres, are alsoprovided. In particular, in one embodiment, a library composed offragments of SATACs are cloned into YACs [yeast artificial chromosomes]that include a detectable marker, such as DNA encoding tyrosinase, andthen introduced into mammalian cells, such as albino mouse embryos. Miceproduced from embryos containing such YACs that include a centromerethat functions in mammals will express the detectable marker. Thus, ifmice are produced from albino mouse embryos into which a functionalmammalian centromere was introduced, the mice will be pigmented or haveregions of pigmentation.

A method for producing repeated tandem arrays of DNA is provided. Thismethod, exemplified herein using telomeric DNA, is applicable to anyrepeat sequence, and in particular, low complexity repeats. The methodprovided herein for synthesis of arrays of tandem DNA repeats are basedin a series of extension steps in which successive doublings of asequence of repeats results in an exponential expansion of the array oftandem repeats. An embodiment of the method of synthesizing DNAfragments containing tandem repeats may generally be described asfollows. Two oligonucleotides are used as starting materials.Oligonucleotide 1 is of length k of repeated sequence (the flanks ofwhich are not relevant) and contains a relatively short stretch (60-90nucleotides) of the repeated sequence, flanked with appropriately chosenrestriction sites:

-   -   5′-S1>>>>>>>>>>>>>>>>>>>>>>>>>>>S2______-3′        where S1 is restriction site 1 cleaved by E1, S2 is a second        restriction site cleaved by E2> represents a simple repeat unit,        and ‘______’ denotes a short (8-10) nucleotide flanking sequence        complementary to oligonucleotide 2:    -   3′-______S3-5′        where S3 is a third restriction site for enzyme E3 and which is        present in the vector to be used during the construction. The        method involves the following steps: (1) oligonucleotides 1 and        2 are annealed; (2) the annealed oligonucleotides are filled-in        to produce a double-stranded (ds) sequence; (3) the        double-stranded DNA is cleaved with restriction enzymes E1 and        E3 and subsequently ligated into a vector (e.g., pUC19 or a        yeast vector) that has been cleaved with the same enzymes E1 and        E3; (4) the insert is isolated from a first portion of the        plasmid by digesting with restriction enzymes E1 and E3, and a        second portion of the plasmid is cut with enzymes E2 (treated to        remove the 3′-overhang) and E3, and the large fragment (plasmid        DNA plus the insert) is isolated; (5) the two DNA fragments (the        S1-S3 insert fragment and the vector plus insert) are ligated;        and (6) steps 4 and 5 are repeated as many times as needed to        achieve the desired repeat sequence size. In each extension        cycle, the repeat sequence size doubles, i.e., if m is the        number of extension cycles, the size of the repeat sequence will        be k×2^(m) nucleotides.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing depicting formation of the MMCneo [theminichromosome] chromosome. A-G represents the successive eventsconsistent with observed data that would lead to the formation andstabilization of the minichromosome.

FIG. 2 shows a schematic summary of the manner in which the observed newchromosomes would form, and the relationships among the different denovo formed chromosomes. In particular, this figure shows a schematicdrawing of the de novo chromosome formation initiated in the centromericregion of mouse chromosome 7. (A) A single E-type amplification in thecentromeric region of chromosome 7 generates a neo-centromere linked tothe integrated “foreign” DNA, and forms a dicentric chromosome. MultipleE-type amplification forms the λ neo-chromosome, which separates fromthe remainder of mouse chromosome 7 through a specific breakage betweenthe centromeres of the dicentric chromosome and which was stabilized ina mouse-hamster hybrid cell line; (B) Specific breakage between thecentromeres of a dicentric chromosome 7 generates a chromosome fragmentwith the neo-centromere, and a chromosome 7 with traces of heterologousDNA at the end; (C) Inverted duplication of the fragment bearing theneo-centromere results in the formation of a stable neo-minichromosome;(D) Integration of exogenous DNA into the heterologous DNA region of theformerly dicentric chromosome 7 initiates H-type amplification, and theformation of a heterochromatic arm. By capturing a euchromatic terminalsegment, this new chromosome arm is stabilized in the form of the“sausage” chromosome; (E) BrdU [5-bromodeoxyuridine] treatment and/ordrug selection induce further H-type amplification, which results in theformation of an unstable gigachromosome: (F) Repeated BrdU treatmentsand/or drug selection induce further H-type amplification including acentromere duplication, which leads to the formation of anotherheterochromatic chromosome arm. It is split off from the chromosome 7 bychromosome breakage, and by acquiring a terminal segment, the stablemegachromosome is formed.

FIG. 3 is a schematic diagram of the replicon structure and a scheme bywhich a megachromosome could be produced.

FIG. 4 sets forth the relationships among some of the exemplary celllines described herein.

FIG. 5 is a diagram of the plasmid pTEMPUD.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

As used herein, a mammalian artificial chromosome [MAC] is a piece ofDNA that can stably replicate and segregate alongside endogenouschromosomes. It has the capacity to accommodate and express heterologousgenes inserted therein. It is referred to as a mammalian artificialchromosome because it includes an active mammalian centromere(s). Plantartificial chromosomes, insect artificial chromosomes and avianartificial chromosomes refer to chromosomes that include plant andinsect centromeres, respectively. A human artificial chromosome [HAC]refers to chromosomes that include human centromeres, BUGACs refer toinsect artificial chromosomes, and AVACs refer to avian artificialchromosomes. Among the MACs provided herein are SATACs, minichromosomes,and in vitro synthesized artificial chromosomes. Methods forconstruction of each type are provided herein.

As used herein, In vitro synthesized artificial chromosomes areartificial chromosomes that is produced by joining the essentialcomponents (at least the centromere, and origins of replication) invitro.

As used herein, endogenous chromosomes refer to genomic chromosomes asfound in the cell prior to generation or introduction of a MAC.

As used herein, stable maintenance of chromosomes occurs when at leastabout 85%, preferably 90%, more preferably 95%, of the cells retain thechromosome. Stability is measured in the presence of a selective agent.Preferably these chromosomes are also maintained in the absence of aselective agent. Stable chromosomes also retain their structure duringcell culturing, suffering neither intrachromosomal nor interchromosomalrearrangements.

As used herein, growth under selective conditions means growth of a cellunder conditions that require expression of a selectable marker forsurvival.

As used herein, an agent that destabilizes a chromosome is any agentknown by those of skill in the art to enhance amplification events,mutations. Such agents, which include BrdU, are well known to those ofskill in the art.

As used herein, de novo with reference to a centromere, refers togeneration of an excess centromere as a result of incorporation of aheterologous DNA fragment using the methods herein.

As used herein, euchromatin and heterochromatin have their recognizedmeanings, euchromatin refers to chromatin that stains diffusely and thattypically contains genes, and heterochromatin refers to chromatin thatremains unusually condensed and that has been thought to betranscriptionally inactive. Highly repetitive DNA sequences [satelliteDNA], at least with respect to mammalian cells, are usually located inregions of the heterochromatin surrounding the centromere [pericentricheterochromatin]. Constitutive heterochromatin refers to heterochromatinthat contains the highly repetitive DNA which is constitutivelycondensed and genetically inactive.

As used herein, BrdU refers to 5-bromodeoxyuridine, which duringreplication is inserted in place of thymidine. BrdU is used as amutagen; it also inhibits condensation of metaphase chromosomes duringcell division.

As used herein, a dicentric chromosome is a chromosome that contains twocentromeres. A multicentric chromosome contains more than twocentromeres.

As used herein, a formerly dicentric chromosome is a chromosome that isproduced when a dicentric chromosome fragments and acquires newtelomeres so that two chromosomes, each having one of the centromeres,are produced. Each of the fragments are replicable chromosomes. If oneof the chromosomes undergoes amplification of euchromatic DNA to producea fully functional chromosome that contains the newly introducedheterologous DNA and primarily [at least more than 50%] euchromatin, itis a minichromosome. The remaining chromosome is a formerly dicentricchromosome. If one of the chromosomes undergoes amplification, wherebyheterochromatin [satellite DNA] is amplified and a euchromatic portion[or arm] remains, it is referred to as a sausage chromosome. Achromosome that is substantially all heterochromatin, except forportions of heterologous DNA, is called a SATAC. Such chromosomes[SATACs] can be produced from sausage chromosomes by culturing the cellcontaining the sausage chromosome under conditions, such as BrdUtreatment and/or growth under selective conditions, that destabilize thechromosome so that a satellite artificial chromosomes [SATAC] isproduced. For purposes herein, it is understood that SATACs may notnecessarily be produced in multiple steps, but may appear after theinitial introduction of the heterologous DNA and growth under selectiveconditions, or they may appear after several cycles of growth underselective conditions and BrdU treatment.

As used herein, a SATAC refers to a chromosome that is substantially allheterochromatin, except for portions of heterologous DNA. Typically,SATACs are satellite DNA based artificial chromosomes, but the termenompasses any chromosome made by the methods herein that contains moreheterochromatin than euchromatin.

As used herein, amplifiable, when used in reference to a chromosome,particularly the method of generating SATACs provided herein, refers toa region of a chromosome that is prone to amplification. Amplifcationtypically occurs during replication and other cellular events involvingrecombination. Such regions are typically regions of the chromosome thatinclude tandem repeats, such as satellite DNA, rDNA and other suchsequences.

As used herein, amplification, with reference to DNA, is a process inwhich segments of DNA are duplicated to yield two or multiple copies ofidentical or nearly identical DNA segments that are typically joined assubstantially tandem or successive repeats or inverted repeats.

As used herein an amplicon is a repeated DNA amplification unit thatcontains a set of inverted repeats of the megareplicon. A megarepliconrepresents a higher order replication unit. For example, with referenceto the SATACs, the megareplicon contains a set of tandem DNA blocks eachcontaining satellite DNA flanked by non-satellite DNA. Contained withinthe megareplicon is a primary replication site, referred to as themegareplicator, which may be involved in organizing and facilitatingreplication of the pericentric heterochromatin and possibly thecentromeres. Within the megareplicon there may be smaller [e.g., 50-300kb in some mammalian cells] secondary replicons. In the exemplifiedSATACS, the megareplicon is defined by two tandem ˜7.5 Mb DNA blocks[see, e.g., FIG. 3]. Within each artificial chromosome [AC] or among apopulation thereof, each amplicon has the same gross structure but maycontain sequence variations. Such variations will arise as a result ofmovement of mobile genetic elements, deletions or insertions ormutations that arise, particularly in culture. Such variation does notaffect the use of the ACs or their overall structure as describedherein.

As used herein, ribosomal RNA [rRNA] is the specialized RNA that formspart of the structure of a ribosome and participates in the synthesis ofproteins. Ribosomal RNA is produced by transcription of genes which, ineukaryotic cells, are present in multiple copies. In human cells, theapproximately 250 copies of rRNA genes per haploid genome are spread outin clusters on at least five different chromosomes (chromosomes 13, 14,15, 21 and 22). In mouse cells, the presence of ribosomal DNA [rDNA] hasbeen verified on at least 11 pairs out of 20 mouse chromosomes[chromosomes 5, 6, 9, 11, 12, 15, 16, 17, 18, 19 and X][see e.g., Roweet al. (1996) Mamm. Genome 7:886-889 and Johnson et al. (1993) Mamm.Genome 4:49-52]. In eukaryotic cells, the multiple copies of the highlyconserved rRNA genes are located in a tandemly arranged series of rDNAunits, which are generally about 40-45 kb in length and contain atranscribed region and a nontranscribed region known as spacer (i.e.,intergenic spacer) DNA which can vary in length and sequence. In thehuman and mouse, these tandem arrays of rDNA units are located adjacentto the pericentric satellite DNA sequences (heterochromatin). Theregions of these chromosomes in which the rDNA is located are referredto as nucleolar organizing regions (NOR) which loop into the nucleolus,the site of ribosome production within the cell nucleus.

As used herein, the minichromosome refers to a chromosome derived from amulticentric, typically dicentric, chromosome [see, e.g., FIG. 1] thatcontains more euchromatic than heterochromatic DNA.

As used herein, a megachromosome refers to a chromosome that, except forintroduced heterologous DNA, is substantially composed ofheterochromatin. Megachromosomes are made of an array of repeatedamplicons that contain two inverted megareplicons bordered by introducedheterologous DNA [see, e.g., FIG. 3 for a schematic drawing of amegachromosome]. For purposes herein, a megachromosome is about 50 to400 Mb, generally about 250-400 Mb. Shorter variants are also referredto as truncated megachromosomes [about 90 to 120 or 150 Mb], dwarfmegachromosomes [˜150-200 Mb] and cell lines, and a micro-megachromosome[˜50-90 Mb, typically 50-60 Mb]. For purposes herein, the termmegachromosome refers to the overall repeated structure based on anarray of repeated chromosomal segments [amplicons] that contain twoinverted megareplicons bordered by any inserted heterologous DNA. Thesize will be specified.

As used herein, genetic therapy involves the transfer or insertion ofheterologous DNA into certain cells, target cells, to produce specificgene products that are involved in correcting or modulating disease. TheDNA is introduced into the selected target cells in a manner such thatthe heterologous DNA is expressed and a product encoded thereby isproduced. Alternatively, the heterologous DNA may in some manner mediateexpression of DNA that encodes the therapeutic product. It may encode aproduct, such as a peptide or RNA, that in some manner mediates,directly or indirectly, expression of a therapeutic product. Genetictherapy may also be used to introduce therapeutic compounds, such asTNF, that are not normally produced in the host or that are not producedin therapeutically effective amounts or at a therapeutically usefultime. Expression of the heterologous DNA by the target cells within anorganism afflicted with the disease thereby enables modulation of thedisease. The heterologous DNA encoding the therapeutic product may bemodified prior to introduction into the cells of the afflicted host inorder to enhance or otherwise alter the product or expression thereof.

As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome in which it is present or which is found in alocation or locations in the genome that differ from that in which itoccurs in nature. It is DNA or RNA that is not endogenous to the celland has been exogenously introduced into the cell. Examples ofheterologous DNA include, but are not limited to, DNA that encodes agene product or gene product(s) of interest, introduced for purposes ofgene therapy or for production of an encoded protein. Other examples ofheterologous DNA include, but are not limited to, DNA that encodestraceable marker proteins, such as a protein that confers drugresistance, DNA that encodes therapeutically effective substances, suchas anti-cancer agents, enzymes and hormones, and DNA that encodes othertypes of proteins, such as antibodies. Antibodies that are encoded byheterologous DNA may be secreted or expressed on the surface of the cellin which the heterologous DNA has been introduced.

As used herein, a therapeutically effective product is a product that isencoded by heterologous DNA that, upon introduction of the DNA into ahost, a product is expressed that effectively ameliorates or eliminatesthe symptoms, manifestations of an inherited or acquired disease or thatcures said disease.

As used herein, transgenic plants refer to plants in which heterologousor foreign DNA is expressed or in which the expression of a genenaturally present in the plant has been altered.

As used herein, operative linkage of heterologous DNA to regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signal sequencesrefers to the relationship between such DNA and such sequences ofnucleotides. For example, operative linkage of heterologous DNA to apromoter refers to the physical relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA in reading frame. Preferred promoters include tissuespecific promoters, such as mammary gland specific promoters, viralpromoters, such TK, CMV, adenovirus promoters, and other promoters knownto those of skill in the art.

As used herein, isolated, substantially pure DNA refers to DNA fragmentspurified according to standard techniques employed by those skilled inthe art, such as that found in Maniatis et al. [(1982) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.].

As used herein, expression refers to the process by which nucleic acidis transcribed into mRNA and translated into peptides, polypeptides, orproteins. If the nucleic acid is derived from genomic DNA, expressionmay, if an appropriate eukaryotic host cell or organism is selected,include splicing of the mRNA.

As used herein, vector or plasmid refers to discrete elements that areused to introduce heterologous DNA into cells for either expression ofthe heterologous DNA or for replication of the cloned heterologous DNA.Selection and use of such vectors and plasmids are well within the levelof skill of the art.

As used herein, transformation/transfection refers to the process bywhich DNA or RNA is introduced into cells. Transfection refers to thetaking up of exogenous nucleic acid, e.g., an expression vector, by ahost cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, by direct uptake using calcium phosphate [CaPO4;see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A.76:1373-1376], polyethylene glycol [PEG]-mediated DNA uptake,electroporation, lipofection [see, e.g., Strauss (1996) Meth. Mol. Biol.54:307-327], microcell fusion [see, EXAMPLES, see, also Lambert (1991)Proc. Natl. Acad. Sci. U.S.A. 88:5907-5911; U.S. Pat. No. 5,396,767,Sawford et al. (1987) Somatic Cell Mol. Genet. 13:279-284; Dhar et al.(1984) Somatic Cell Mol. Genet. 10:547-559; and McNeill-Killary et al.(1995) Meth. Enzymol. 254:133-152], lipid-mediated carrier systems [see,e.g., Teifel et al. (1995) Biotechniques 19:79-80; Albrecht et al.(1996) Ann. Hematol. 72:73-79; Holmen et al. (1995) In Vitro Cell Dev.Biol. Anim. 31:347-351; REmy et al. (1994) Bioconjug. Chem. 5:647-654;Le Bolch et al. (1995) Tetrahedron Lett. 36:6681-6684; Loeffler et al.(1993) Meth. Enzymol. 217:599-618] or other suitable method. Successfultransfection is generally recognized by detection of the presence of theheterologous nucleic acid within the transfected cell, such as anyindication of the operation of a vector within the host cell.Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegration.

As used herein, injected refers to the microinjection [use of a smallsyringe] of DNA into a cell.

As used herein, substantially homologous DNA refers to DNA that includesa sequence of nucleotides that is sufficiently similar to another suchsequence to form stable hybrids under specified conditions.

It is well known to those of skill in this art that nucleic acidfragments with different sequences may, under the same conditions,hybridize detectably to the same “target” nucleic acid. Two nucleic acidfragments hybridize detectably, under stringent conditions over asufficiently long hybridization period, because one fragment contains asegment of at least about 14 nucleotides in a sequence which iscomplementary [or nearly complementary] to the sequence of at least onesegment in the other nucleic acid fragment. If the time during whichhybridization is allowed to occur is held constant, at a value duringwhich, under preselected stringency conditions, two nucleic acidfragments with exactly complementary base-pairing segments hybridizedetectably to each other, departures from exact complementarity can beintroduced into the base-pairing segments, and base-pairing willnonetheless occur to an extent sufficient to make hybridizationdetectable. As the departure from complementarity between thebase-pairing segments of two nucleic acids becomes larger, and asconditions of the hybridization become more stringent, the probabilitydecreases that the two segments will hybridize detectably to each other.

Two single-stranded nucleic acid segments have “substantially the samesequence,” within the meaning of the present specification, if (a) bothform a base-paired duplex with the same segment, and (b) the meltingtemperatures of said two duplexes in a solution of 0.5×SSPE differ byless than 10° C. If the segments being compared have the same number ofbases, then to have “substantially the same sequence”, they willtypically differ in their sequences at fewer than 1 base in 10. Methodsfor determining melting temperatures of nucleic acid duplexes are wellknown [see, e.g., Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284and references cited therein].

As used herein, a nucleic acid probe is a DNA or RNA fragment thatincludes a sufficient number of nucleotides to specifically hybridize toDNA or RNA that includes identical or closely related sequences ofnucleotides. A probe may contain any number of nucleotides, from as fewas about 10 and as many as hundreds of thousands of nucleotides. Theconditions and protocols for such hybridization reactions are well knownto those of skill in the art as are the effects of probe size,temperature, degree of mismatch, salt concentration and other parameterson the hybridization reaction. For example, the lower the temperatureand higher the salt concentration at which the hybridization reaction iscarried out, the greater the degree of mismatch that may be present inthe hybrid molecules.

To be used as a hybridization probe, the nucleic acid is generallyrendered detectable by labelling it with a detectable moiety or label,such as ³²P, ³H and ¹⁴C, or by other means, including chemicallabelling, such as by nick-translation in the presence of deoxyuridylatebiotinylated at the 5′-position of the uracil moiety. The resultingprobe includes the biotinylated uridylate in place of thymidylateresidues and can be detected [via the biotin moieties] by any of anumber of commercially available detection systems based on binding ofstreptavidin to the biotin. Such commercially available detectionsystems can be obtained, for example, from Enzo Biochemicals, Inc. [NewYork, N.Y.]. Any other label known to those of skill in the art,including non-radioactive labels, may be used as long as it renders theprobes sufficiently detectable, which is a function of the sensitivityof the assay, the time available [for culturing cells, extracting DNA,and hybridization assays], the quantity of DNA or RNA available as asource of the probe, the particular label and the means used to detectthe label.

Once sequences with a sufficiently high degree of homology to the probeare identified, they can readily be isolated by standard techniques,which are described, for example, by Maniatis et al. ((1982) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

As used herein, conditions under which DNA molecules form stable hybridsand are considered substantially homologous are such that DNA moleculeswith at least about 60% complementarity form stable hybrids. Such DNAfragments are herein considered to be “substantially homologous”. Forexample, DNA that encodes a particular protein is substantiallyhomologous to another DNA fragment if the DNA forms stable hybrids suchthat the sequences of the fragments are at least about 60% complementaryand if a protein encoded by the DNA retains its activity.

For purposes herein, the following stringency conditions are defined:

-   -   1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.    -   2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.    -   3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.        or any combination of salt and temperature and other reagents        that result in selection of the same degree of mismatch or        matching.

As used herein, immunoprotective refers to the ability of a vaccine orexposure to an antigen or immunity-inducing agent, to confer upon a hostto whom the vaccine or antigen is administered or introduced, theability to resist infection by a disease-causing pathogen or to havereduced symptoms. The selected antigen is typically an antigen that ispresented by the pathogen.

As used herein, all assays and procedures, such as hybridizationreactions and antibody-antigen reactions, unless otherwise specified,are conducted under conditions recognized by those of skill in the artas standard conditions.

A. Preparation of Cell Lines Containing MACs

1. The Megareplicon

The methods, cells and MACs provided herein are produced by virtue ofthe discovery of the existence of a higher-order replication unit[megareplicon] of the centromeric region. This megareplicon is delimitedby a primary replication initiation site [megareplicator], and appearsto facilitate replication of the centromeric heterochromatin, and mostlikely, centromeres. Integration of heterologous DNA into themegareplicator region or in close proximity thereto, initiates alarge-scale amplification of megabase-size chromosomal segments, whichleads to de novo chromosome formation in living cells.

DNA sequences that provide a preferred megareplicator are the rDNA unitsthat give rise to ribosomal RNA (rRNA). In mammals, particularly miceand humans, these rDNA units contain specialized elements, such as theorigin of replication (or origin of bidirectional replication, i.e.,OBR, in mouse) and amplification promoting sequences (APS) andamplification control elements (ACE) (see, e.g., Gogel et al. (1996)Chromosoma 104:511-518; Coffman et al. (1993) Exp. Cell. Res.209:123-132; Little et al. (1993) Mol. Cell. Biol. 13:6600-6613; Yoon etal. (1995) Mol. Cell. Biol. 15:2482-2489; Gonzalez and Sylvester (1995)Genomics 27:320-328; Miesfeld and Arnheim (1982) Nuc. Acids Res.10:3933-3949]); Maden et al. (1987) Biochem. J. 246:519-527).

As described herein, without being bound by any theory, thesespecialized elements may facilitate replication and/or amplification ofmegabase-size chromosomal segments in the de novo formation ofchromosomes, such as those described herein, in cells. These specializedelements are typically located in the nontranscribed intergenic spacerregion upstream of the transcribed region of rDNA. The intergenic spacerregion may itself contain internally repeated sequences which can beclassified as tandemly repeated blocks and nontandem blocks (see e.g.,Gonzalez and Sylvester (1995) Genomics 27:320-328). In mouse rDNA, anorigin of bidirectional replication may be found within a 3-kbinitiation zone centered approximately 1.6 kb upstream of thetranscription start site (see, e.g., Gogel et al. (1996) Chromosoma104:511-518). The sequences of these specialized elements tend to havean altered chromatin structure, which may be detected, for example, bynuclease hypersensitivity or the presence of AT-rich regions that cangive rise to bent DNA structures. An exemplary sequence encompassing anorigin of replication is shown in SEQ ID NO. 16 and in GENBANK accessionno. X82564 at about positions 2430-5435. Exemplary sequencesencompassing amplification-promoting sequences include nucleotides690-1060 and 1105-1530 of SEQ ID NO. 16.

In human rDNA, a primary replication initiation site may be found a fewkilobase pairs upstream of the transcribed region and secondaryinitiation sites may be found throughout the nontranscribed intergenicspacer region (see, e.g., Yoon et al. (1995) Mol. Cell. Biol.15:2482-2489). A complete human rDNA repeat unit is presented in GENBANKas accession no. U13369 and is set forth in SEQ ID NO. 17 herein.Another exemplary sequence encompassing a replication initiation sitemay be found within the sequence of nucleotides 35355-42486 in SEQ IDNO. 17 particularly within the sequence of nucleotides 37912-42486 andmore particularly within the sequence of nucleotides 37912-39288 of SEQID NO. 17 (see Coffman et al. (1993) Exp. Cell. Res. 209:123-132).

Cell lines containing MACs can be prepared by transforming cells,preferably a stable cell line, with a heterologous DNA fragment thatencodes a selectable marker, culturing under selective conditions, andidentifying cells that have a multicentric, typically dicentric,chromosome. These cells can then be manipulated as described herein toproduce the minichromosomes and other MACs, particularly theheterochromatic SATACs, as described herein.

Development of a multicentric, particularly dicentric, chromosometypically is effected through integration of the heterologous DNA in thepericentric heterochromatin, preferably in the centromeric regions ofchromosomes carrying rDNA sequences. Thus, the frequency ofincorporation can be increased by targeting to these regions, such as byincluding DNA, including, but not limited to, rDNA or satellite DNA, inthe heterologous fragment that encodes the selectable marker. Among thepreferred targeting sequences for directing the heterologous DNA to thepericentromeric heterochromatin are rDNA sequences that targetcentromeric regions of chromosomes that carry rRNA genes. Such sequencesinclude, but are not limited to, the DNA of SEQ ID NO. 16 and GENBANKaccession no. X82564 and portions thereof, the DNA of SEQ ID NO. 17 andGENBANK accession no. U13369 and portions thereof, and the DNA of SEQ IDNOS. 18-24. A particular vector incorporating from within SEQ ID NO. 16for use in directing integration of heterologous DNA into chromosomalrDNA is pTERPUD (see Example 12). Satellite DNA sequences can also beused to direct the heterologous DNA to integrate into the pericentricheterochromatin. For example, vectors pTEMPUD and pHASPUD, which containmouse and human satellite DNA, respectively, are provided herein (seeExample 12) as exemplary vectors for introduction of heterologous DNAinto cells for de novo artificial chromosome formation.

The resulting cell lines can then be treated as the exemplified cellsherein to produce cells in which the dicentric chromosome hasfragmented. The cells can then be used to introduce additional selectivemarkers into the fragmented dicentric chromosome (i.e., formerlydicentric chromosome), whereby amplification of the pericentricheterochromatin will produce the heterochromatic chromosomes.

The following discussion describes this process with reference to theEC3/7 line and the resulting cells. The same procedures can be appliedto any other cells, particularly cell lines to create SATACs andeuchromatic minichromosomes.

2. Formation of de novo Chromosomes

De novo centromere formation in a transformed mouse LMTK-fibro-blastcell line [EC3/7] after cointegration of λ constructs [λCM8 andλgtWESneo] carrying human and bacterial DNA [Hadlaczky et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110 and U.S. application Ser. No.08/375,271] has been shown. The integration of the “heterologous”engineered human, bacterial and phage DNA, and the subsequentamplification of mouse and heterologous DNA that led to the formation ofa dicentric chromosome, occurred at the centromeric region of the shortarm of a mouse chromosome. By G-banding, this chromosome was identifiedas mouse chromosome 7. Because of the presence of two functionallyactive centromeres on the same chromosome, regular breakages occurbetween the centromeres. Such specific chromosome breakages gave rise tothe appearance [in approximately 10% of the cells] of a chromosomefragment carrying the neo-centromere. From the EC3/7 cell line [see,U.S. Pat. No. 5,288,625, deposited at the European Collection of AnimalCell Culture (hereinafter ECACC) under accession no. 90051001; see, alsoHadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110, andU.S. application Ser. No. 08/375,271 and the corresponding publishedEuropean application EP 0 473 253, two sublines [EC3/7C5 and EC3/7C6]were selected by repeated single-cell cloning. In these cell lines, theneo-centromere was found exclusively on a minichromosome[neo-minichromosome], while the formerly dicentric chromosome carriedtraces of “heterologous” DNA.

It has now been discovered that integration of DNA encoding a selectablemarker in the heterochromatic region of the centromere led to formationof the dicentric chromosome.

3. The Neo-Minichromosome

The chromosome breakage in the EC3/7 cells, which separates theneo-centromere from the mouse chromosome, occurred in the G-bandpositive “heterologous” DNA region. This is supported by the observationof traces of λ and human DNA sequences at the broken end of the formerlydicentric chromosome. Comparing the G-band pattern of the chromosomefragment carrying the neo-centromere with that of the stableneo-minichromosome, it is apparent that the neo-minichromosome is aninverted duplicate of the chromosome fragment that bears theneo-centromere. This is supported by the observation that although theneo-minichromosome carries only one functional centromere, both ends ofthe minichromosome are heterochromatic, and mouse satellite DNAsequences were found in these heterochromatic regions by in situhybridization.

Mouse cells containing the minichromosome, which contains multiplerepeats of the heterologous DNA, which in the exemplified embodiment isλ DNA and the neomycin-resistance gene, can be used as recipient cellsin cell transformation. Donor DNA, such as selected heterologous DNAcontaining λ DNA linked to a second selectable marker, such as the geneencoding hygromycin phosphotransferase which confers hygromycinresistance [hyg], can be introduced into the mouse cells and integratedinto the minichromosomes by homologous recombination of λ DNA in thedonor DNA with that in the minichromosomes. Integration is verified byin situ hybridization and Southern blot analyses. Transcription andtranslation of the heterologous DNA is confirmed by primer extension andimmunoblot analyses.

For example, DNA has been targeted into the neo-minichromosome inEC3/7C5 cells using a λ DNA-containing construct [pNem1ruc] that alsocontains DNA encoding hygromycin resistance and the Renilla luciferasegene linked to a promoter, such as the cytomegalovirus [CMV] earlypromoter, and the bacterial neomycin resistance-encoding DNA.Integration of the donor DNA into the chromosome in selected cells[designated PHN4] was confirmed by nucleic acid amplification [PCR] andin situ hybridization. Events that would produce a neo-minichromosomeare depicted in FIG. 1.

The resulting engineered minichromosome that contains the heterologousDNA can then be transferred by cell fusion into a recipient cell line,such as Chinese hamster ovary cells [CHO] and correct expression of theheterologous DNA can be verified. Following production of the cells,metaphase chromosomes are obtained, such as by addition of colchicine,and the chromosomes purified by addition of AT- and GC-specific dyes ona dual laser beam based cell sorter (see Example 10 B for a descriptionof methods of isolating artificial chromomsomes). Preparative amounts ofchromosomes [5×10⁴−5×10⁷ chromosomes/ml] at a purity of 95% or highercan be obtained. The resulting chromosomes are used for delivery tocells by methods such as microinjection and liposome-mediated transfer.

Thus, the neo-minichromosome is stably maintained in cells, replicatesautonomously, and permits the persistent long-term expression of the neogene under non-selective culture conditions. It also contains megabasesof heterologous known DNA [λ DNA in the exemplified embodiments] thatserves as target sites for homologous recombination and integration ofDNA of interest. The neo-minichromosome is, thus, a vector for geneticengineering of cells. It has been introduced into SCID mice, and shownto replicate in the same manner as endogenous chromosomes.

The methods herein provide means to induce the events that lead toformation of the neo-minichromosome by introducing heterologous DNA witha selective marker [preferably a dominant selectable marker] into cellsand culturing the cells under selective conditions. As a result, cellsthat contain a multicentric, e.g., dicentric chromosome, or fragmentsthereof, generated by amplification are produced. Cells with thedicentric chromosome can then be treated to destabilize the chromosomeswith agents, such as BrdU and/or culturing under selective conditions,resulting in cells in which the dicentric chromosome has formed twochromosomes, a so-called minichromosome, and a formerly dicentricchromosome that has typically undergone amplification in theheterochromatin where the heterologous DNA has integrated to produce aSATAC or a sausage chromosome [discussed below]. These cells can befused with other cells to separate the minichromosome from the formerlydicentric chromosome into different cells so that each type of MAC canbe manipulated separately.

4. Preparation of SATACs

An exemplary protocol for preparation of SATACs is illustrated in FIG. 2[particularly D, E and F] and FIG. 3 [see, also the EXAMPLES,particularly EXAMPLES 4-7].

To prepare a SATAC, the starting materials are cells, preferably astable cell line, such as a fibroblast cell line, and a DNA fragmentthat includes DNA that encodes a selective marker. The DNA fragment isintroduced into the cell by methods of DNA transfer, including but notlimited to direct uptake using calcium phosphate, electroporation, andlipid-mediated transfer. To insure integration of the DNA fragment inthe heterochromatin, it is preferable to start with DNA that will betargeted to the pericentric heterochromatic region of the chromosome,such as λCM8 and vectors provided herein, such as pTEMPUD [FIG. 5] andpHASPUD (see Example 12) that include satellite DNA, or specificallyinto rDNA in the centromeric regions of chromosomes containing rDNAsequences. After introduction of the DNA, the cells are grown underselective conditions. The resulting cells are examined and any that havemulticentric, particularly dicentric, chromosomes [or heterochromaticchromosomes or sausage chromosomes or other such structure; see, FIG.2D, 2E and 2F] are selected.

In particular, if a cell with a dicentric chromosome is selected, it canbe grown under selective conditions, or, preferably, additional DNAencoding a second selectable marker is introduced, and the cells grownunder conditions selective for the second marker. The resulting cellsshould include chromosomes that have structures similar to thosedepicted in FIGS. 2D, 2E, 2F. Cells with a structure, such as thesausage chromosome, FIG. 2D, can be selected and fused with a secondcell line to eliminate other chromosomes that are not of interest. Ifdesired, cells with other chromosomes can be selected and treated asdescribed herein. If a cell with a sausage chromosome is selected, itcan be treated with an agent, such as BrdU, that destabilizes thechromosome so that the heterochromatic arm forms a chromosome that issubstantially heterochromatic [i.e., a megachromosome, see, FIG. 2F].Structures such as the gigachromsome in which the heterochromatic armhas amplified but not broken off from the euchromatic arm, will also beobserved. The megachromosome is a stable chromosome. Furthermanipulation, such as fusions and growth in selective conditions and/orBrdU treatment or other such treatment, can lead to fragmentation of themegachromosome to form smaller chromosomes that have the amplicon as thebasic repeating unit.

The megachromosome can be further fragmented in vivo using a chromosomefragmentation vector, such as pTEMPUD [see, FIG. 5 and EXAMPLE 12],pHASPUD or pTERPUD (see Example 12) to ultimately produce a chromosomethat comprises a smaller stable replicable unit, about 15 Mb-60 Mb,containing one to four megareplicons.

Thus, the stable chromosomes formed de novo that originate from theshort arm of mouse chromosome 7 have been analyzed. This chromosomeregion shows a capacity for amplification of large chromosome segments,and promotes de novo chromosome formation. Large-scale amplification atthe same chromosome region leads to the formation of dicentric andmulticentric chromosomes, a minichromosome, the 150-200 Mb size λneo-chromosome, the “sausage” chromosome, the 500-1000 Mbgigachromosome, and the stable 250-400 Mb megachromosome.

A clear segmentation is observed along the arms of the megachromosome,and analyses show that the building units of this chromosome areamplicons of ˜30 Mb composed of mouse major satellite DNA with theintegrated “foreign” DNA sequences at both ends. The ˜30 Mb ampliconsare composed of two ˜15 Mb inverted doublets of ˜7.5 Mb mouse majorsatellite DNA blocks, which are separated from each other by a narrowband of non-satellite sequences [see, e.g., FIG. 3]. The widernon-satellite regions at the amplicon borders contain integrated,exogenous [heterologous] DNA, while the narrow bands of non-satelliteDNA sequences within the amplicons are integral parts of the pericentricheterochromatin of mouse chromosomes. These results indicate that the˜7.5 Mb blocks flanked by non-satellite DNA are the building units ofthe pericentric heterochromatin of mouse chromosomes, and the ˜15 Mbsize pericentric regions of mouse chromosomes contain two ˜7.5 Mb units.

Apart from the euchromatic terminal segments, the whole megachromosomeis heterochromatic, and has structural homogeneity. Therefore, thislarge chromosome offers a unique possibility for obtaining informationabout the amplification process, and for analyzing some basiccharacteristics of the pericentric constitutive heterochromatin, as avector for heterologous DNA, and as a target for further fragmentation.

As shown herein, this phenomenon is generalizable and can be observedwith other chromosomes. Also, although these de novo formed chromosomesegments and chromosomes appear different, there are similarities thatindicate that a similar amplification mechanism plays a role in theirformation: (i) in each case, the amplification is initiated in thecentromeric region of the mouse chromosomes and large (Mb size)amplicons are formed; (ii) mouse major satellite DNA sequences areconstant constituents of the amplicons, either by providing the bulk ofthe heterochromatic amplicons [H-type amplification], or by borderingthe aeuchromatic amplicons [E-type amplification]; (iii) formation ofinverted segments can be demonstrated in the λ neo-chromosome andmegachromosome; (iv) chromosome arms and chromosomes formed by theamplification are stable and functional.

The presence of inverted chromosome segments seems to be a commonphenomenon in the chromosomes formed de novo at the centromeric regionof mouse chromosome 7. During the formation of the neo-minichromosome,the event leading to the stabilization of the distal segment of mousechromosome 7 that bears the neo-centromere may have been the formationof its inverted duplicate. Amplicons of the megachromosome are inverteddoublets of ˜7.5 Mb mouse major satellite DNA blocks.

5. Cell Lines

Cell lines that contain MACs, such as the minichromosome, the λ-neochromosome, and the SATACs are provided herein or can be produced by themethods herein. Such cell lines provide a convenient source of thesechromosomes and can be manipulated, such as by cell fusion or productionof microcells for fusion with selected cell lines, to deliver thechromosome of interest into hybrid cell lines. Exemplary cell lines aredescribed herein and some have been deposited with the ECACC.

a. EC3/7C5 and EC3/7C6

Cell lines EC3/7C5 and EC3/7C6 were produced by single cell cloning ofEC3/7. For exemplary purposes EC3/7C5 has been deposited with the ECACC.These cell lines contain a minichromosome and the formerly dicentricchromosome from EC3/7. The stable minichromosomes in cell lines EC3/7C5and EC3/7C6 appear to be the same and they seem to be duplicatedderivatives of the ˜10-15 Mb “broken-off” fragment of the dicentricchromosome. Their similar size in these independently generated celllines might indicate that ˜20-30 Mb is the minimal or close to theminimal physical size for a stable minichromosome.

b. TF1004G19

Introduction of additional heterologous DNA, including DNA encoding asecond selectable marker, hygromycin phosphotransferase, i.e., thehygromycin-resistance gene, and also a detectable marker,β-galactosidase (i.e., encoded by the lacZ gene), into the EC3/7C5 cellline and growth under selective conditions produced cells designatedTF1004G19. In particular, this cell line was produced from the EC3/7C5cell line by cotransfection with plasmids pH132, which contains ananti-HIV ribozyme and hygromycin-resistance gene, pCH110 [encodesβ-galactosidase] and λ phage [λcl 875 Sam 7] DNA and selection withhygromycin B.

Detailed analysis of the TF1004G19 cell line by in situ hybridizationwith λ phage and plasmid DNA sequences revealed the formation of thesausage chromosome. The formerly dicentric chromosome of the EC3/7C5cell line translocated to the end of another acrocentric chromosome. Theheterologous DNA integrated into the pericentric heterochromatin of theformerly dicentric chromosome and is amplified several times withmegabases of mouse pericentric heterochromatic satellite DNA sequences[FIG. 2D] forming the “sausage” chromosome. Subsequently the acrocentricmouse chromosome was substituted by a euchromatic telomere.

In situ hybridization with biotin-labeled subfragments of thehygromycin-resistance and β-galactosidase genes resulted in ahybridization signal only in the heterochromatic arm of the sausagechromosome, indicating that in TF1004G19 transformant cells these genesare localized in the pericentric heterochromatin.

A high level of gene expression, however, was detected. In general,heterochromatin has a silencing effect in Drosophila, yeast and on theHSV-tk gene introduced into satellite DNA at the mouse centromere. Thus,it was of interest to study the TF1004G19 transformed cell line toconfirm that genes located in the heterochromatin were indeed expressed,contrary to recognized dogma.

For this purpose, subclones of TF1004G19, containing a different sausagechromosome [see FIG. 2D], were established by single cell cloning.Southern hybridization of DNA isolated from the subclones withsubfragments of hygromycin phosphotransferase and lacZ genes showed aclose correlation between the intensity of hybridization and the lengthof the sausage chromosome. This finding supports the conclusion thatthese genes are localized in the heterochromatic arm of the sausagechromosome.

(1) TF1004G-19C5

TF1004G-19C5 is a mouse LMTK fibroblast cell line containingneo-minichromosomes and stable “sausage” chromosomes. It is a subcloneof TF1004G19 and was generated by single-cell cloning of the TF1004G19cell line. It has been deposited with the ECACC as an exemplary cellline and exemplary source of a sausage chromosome. Subsequent fusion ofthis cell line with CHO K20 cells and selection with hygromycin and G418and HAT (hypoxanthine, aminopteria, and thymidine medium; see Szybalskiet al. (1962) Proc. Natl. Acad. Sci. 48:2026) resulted in hybrid cells(designated 19C5×Ha4) that carry the sausage chromosome and theneo-minichromosome. BrdU treatment of the hybrid cells, followed bysingle cell cloning and selection with G418 and/or hygromycin producedvarious cells that carry chromosomes of interest, including GB43 andG3D5.

(2) Other Subclones

Cell lines GB43 and G3D5 were obtained by treating 19C5×Ha4 cells withBrdU followed by growth in G418-containing selective medium andretreatment with BrdU. The two cell lines were isolated by single cellcloning of the selected cells. GB43 cells contain the neo-minichromosomeonly. G3D5, which has been deposited with the ECACC, carries theneo-minichromosome and the megachromosome. Single cell cloning of thiscell line followed by growth of the subclones in G418- andhygromycin-containing medium yielded subclones such as the GHB42 cellline carrying the neo-minichromosome and the megachromosome. H1D3 is amouse-hamster hybrid cell line carrying the megachromosome, but noneo-minichromosome, and was generated by treating 19C5×Ha4 cells withBrdU followed by growth in hygromycin-containing selective medium andsingle cell subcloning of selected cells. Fusion of this cell line withthe CD4⁺ HeLa cell line that also carries DNA encoding an additionalselection gene, the neomycin-resistance gene, produced cells [designatedH1×HE41 cells] that carry the megachromosome as well as a humanchromosome that carries CD4neo. Further BrdU treatment and single cellcloning produced cell lines, such as 1B3, that include cells with atruncated megachromosome.

5. DNA Constructs Used to Transform the Cells

Heterologous DNA can be introduced into the cells by transfection orother suitable method at any stage during preparation of the chromosomes[see, e.g., FIG. 4]. In general, incorporation of such DNA into the MACsis assured through site-directed integration, such as may beaccomplished by inclusion of λ-DNA in the heterologous DNA (for theexemplified chromosomes), and also an additional selective marker gene.For example, cells containing a MAC, such as the minichromosome or aSATAC, can be cotransfected with a plasmid carrying the desiredheterologous DNA, such as DNA encoding an HIV ribozyme, the cysticfibrosis gene, and DNA encoding a second selectable marker, such ashygromycin resistance. Selective pressure is then applied to the cellsby exposing them to an agent that is harmful to cells that do notexpress the new selectable marker. In this manner, cells that includethe heterologous DNA in the MAC are identified. Fusion with a secondcell line can provide a means to produce cell lines that contain oneparticular type of chromosomal structure or MAC.

Various vectors for this purpose are provided herein [see, Examples] andothers can be readily constructed. The vectors preferably include DNAthat is homologous to DNA contained within a MAC in order to target theDNA to the MAC for integration therein. The vectors also include aselectable marker gene and the selected heterologous gene(s) ofinterest. Based on the disclosure herein and the knowledge of theskilled artisan, one of skill can construct such vectors.

Of particular interest herein is the vector pTEMPUD and derivativesthereof that can target DNA into the heterochromatic region of selectedchromosomes. These vectors can also serve as fragmentation vectors [see,e.g., Example 12].

Heterologous genes of interest include any gene that encodes atherapeutic product and DNA encoding gene products of interest. Thesegenes and DNA include, but are not limited to: the cystic fibrosis gene[CF], the cystic fibrosis transmembrane regulator (CFTR) gene [see,e.g., U.S. Pat. No. 5,240,846; Rosenfeld et al. (1992) Cell 68:143-155;Hyde et al. (1993) Nature 362: 250-255; Kerem et al. (1989) Science245:1073-1080; Riordan et al.(1989) Science 245:1066-1072; Rommens etal. (1989) Science 245:1059-1065; Osborne et al. (1991) Am. J. Hum.Genetics 48:6089-6122; White et al. (1990) Nature 344:665-667; Dean etal. (1990) Cell 61:863-870; Erlich et al. (1991) Science 252:1643; andU.S. Pat. Nos. 5,453,357, 5,449,604, 5,434,086, and 5,240,846, whichprovides a retroviral vector encoding the normal CFTR gene].

B. Isolation of Artificial Chromosomes

The MACs provided herein can be isolated by any suitable method known tothose of skill in the art. Also, methods are provided herein foreffecting substantial purification, particularly of the SATACs. SATACshave been isolated by fluorescence-activated cell sorting [FACS]. Thismethod takes advantage of the nucleotide base content of the SATACs,which, by virtue of their high heterochromatic DNA content, will differfrom any other chromosomes in a cell. In particular embodiment,metaphase chromosomes are isolated and stained with base-specific dyes,such as Hoechst 33258 and chromomycin A3. Fluorescence-activated cellsorting will separate the SATACs from the endogenous chromosomes. Adual-laser cell sorter [FACS Vantage Becton Dickinson ImmunocytometrySystems] in which two lasers were set to excite the dyes separately,allowed a bivariate analysis of the chromosomes by base-pair compositionand size. Cells containing such SATACs can be similarly sorted.

Additional methods provided herein for isolation of artificialchromosomes from endogenous chromosomes include procedures that areparticularly well suited for large-scale isolation of artificialchromosomes such as SATACs. In these methods, the size and densitydifferences between SATACs and endogenous chromosomes are exploited toeffect separation of these two types of chromosomes. Such methodsinvolve techniques such as swinging bucket centrifugation, zonal rotorcentrifugation, and velocity sedimentation. Affinity-, particularlyimmunoaffinity-, based methods for separation of artificial fromendogenous chromosomes are also provided herein. For example, SATACs,which are predominantly heterochromatin, may be separated fromendogenous chromosomes through immunoaffinity procedures involvingantibodies that specifically recognize heterochromatin, and/or theproteins associated therewith, when the endogenous chromosomes containrelatively little heterochromatin, such as in hamster cells.

C. In vitro Construction of Artificial Chromosomes

Artificial chromosomes can be constructed in vitro by assembling thestructural and functional elements that contribute to a completechromosome capable of stable replication and segregation alongsideendogenous chromosomes in cells. The identification of the discreteelements that in combination yield a functional chromosome has madepossible the in vitro generation of artificial chromosomes. The processof in vitro construction of artificial chromosomes, which can be rigidlycontrolled, provides advantages that may be desired in the generation ofchromosomes that, for example, are required in large amounts or that areintended for specific use in transgenic animal systems.

For example, in vitro construction may be advantageous when efficiencyof time and scale are important considerations in the preparation ofartificial chromosomes. Because in vitro construction methods do notinvolve extensive cell culture procedures, they may be utilized when thetime and labor required to transform, feed, cultivate, and harvest cellsused in in vivo cell-based production systems is unavailable.

In vitro construction may also be rigorously controlled with respect tothe exact manner in which the several elements of the desired artificialchromosome are combined and in what sequence and proportions they areassembled to yield a chromosome of precise specifications. These aspectsmay be of significance in the production of artificial chromosomes thatwill be used in live animals where it is desirable to be certain thatonly very pure and specific DNA sequences in specific amounts are beingintroduced into the host animal.

The following describes the processes involved in the construction ofartificial chromosomes in vitro, utilizing a megachromosome as exemplarystarting material.

1. Identification and Isolation of the Components of the ArtificialChromosome

The MACs provided herein, particularly the SATACs, are elegantly simplechromosomes for use in the identification and isolation of components tobe used in the in vitro construction of artificial chromosomes. Theability to purify MACs to a very high level of purity, as describedherein, facilitates their use for these purposes. For example, themegachromosome, particularly truncated forms thereof [i.e. cell lines,such as 1B3 and mM2C1, which are derived from H1D3 (deposited at theEuropean Collection of Animal Cell Culture (ECACC) under Accession No.96040929, see EXAMPLES below) serve as starting materials.

For example, the mM2C1 cell line contains a micro-megachromosome (˜50-60kB), which advantageously contains only one centromere, two regions ofintegrated heterologous DNA with adjacent rDNA sequences, with theremainder of the chromosomal DNA being mouse major satellite DNA. Othertruncated megachromosomes can serve as a source of telomeres, ortelomeres can be provided (see, Examples below regarding construction ofplasmids containing tandemly repeated telomeric sequences). Thecentromere of the mM2C1 cell line contains mouse minor satellite DNA,which provides a useful tag for isolation of the centromeric DNA.

Additional features of particular SATACs provided herein, such as themicro-megachromosome of the mM2C1 cell line, that make them uniquelysuited to serve as starting materials in the isolation andidentification of chromosomal components include the fact that thecentromeres of each megachromosome within a single specific cell lineare identical. The ability to begin with a homogeneous centromere source(as opposed to a mixture of different chromosomes having differingcentromeric sequences) greatly facilitates the cloning of the centromereDNA. By digesting purified megachromosomes, particularly truncatedmegachromosomes, such as the micro-megachromosome, with appropriaterestriction endonucleases and cloning the fragments into thecommercially available and well known YAC vectors (see, e.g., Burke etal. (1987) Science 236:806-812), BAC vectors (see, e.g., Shizuya et al.(1992) Proc. Natl. Acad. Sci. U.S.A. 89: 8794-8797 bacterial artificialchromosomes which have a capacity of incorporating 0.9-1 Mb of DNA) orPAC vectors (the P1 artificial chromosome vector which is a P1 plasmidderivative that has a capacity of incorporating 300 kb of DNA and thatis delivered to E. coli host cells by electroporation rather than bybacteriophage packaging; see, e.g., loannou et al. (1994) NatureGenetics 6:84-89; Pierce et al. (1992) Meth. Enzymol. 216:549-574;Pierce et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:2056-2060; U.S.Pat. No. 5,300,431 and International PCT application No. WO 92/14819)vectors, it is possible for as few as 50 clones to represent the entiremicro-megachromosome.

a. Centromeres

An exemplary centromere for use in the construction of a mammalianartificial chromosome is that contained within the megachromosome of anyof the megachromosome-containing cell lines provided herein, such as,for example, H1D3 and derivatives thereof, such as mM2C1 cells.Megachromosomes are isolated from such cell lines utilizing, forexample, the procedures described herein, and the centromeric sequenceis extracted from the isolated megachromosomes. For example, themegachromosomes may be separated into fragments utilizing selectedrestriction endonucleases that recognize and cut at sites that, forinstance, are primarily located in the replication and/or heterologousDNA integration sites and/or in the satellite DNA. Based on the sizes ofthe resulting fragments, certain undesired elements may be separatedfrom the centromere-containing sequences. The centromere-containing DNA,which could be as large as 1 Mb.

Probes that specifically recognize the centromeric sequences, such asmouse minor satellite DNA-based probes [see, e.g., Wong et al. (1988)Nucl. Acids Res. 16:11645-11661], may be used to isolate thecentromere-containing YAC, BAC or PAC clones derived from themegachromosome. Alternatively, or in conjunction with the directidentification of centromere-containing megachromosomal DNA, probes thatspecifically recognize the non-centromeric elements, such as probesspecific for mouse major satellite DNA, the heterologous DNA and/orrDNA, may be used to identify and eliminate the non-centromericDNA-containing clones.

Additionally, centromere cloning methods described herein may beutilized to isolate the centromere-containing sequence of themegachromosome. For example, Example 12 describes the use of YAC vectorsin combination with the murine tyrosinase gene and NMRI/Han mice foridentification of the centromeric sequence.

Once the centromere fragment has been isolated, it may be sequenced andthe sequence information may in turn be used in PCR amplification ofcentromere sequences from megachromosomes or other sources ofcentromeres. Isolated centromeres may also be tested for function invivo by transferring the DNA into a host mammalian cell. Functionalanalysis may include, for example, examining the ability of thecentromere sequence to bind centromere-binding proteins. The clonedcentromere will be transferred to mammalian cells with a selectablemarker gene and the binding of a centromere-specific protein, such asanti-centromere antibodies (e.g., LU85 1, see, Hadlaczky et al. (1986)Exp. Cell Res. 167:1-15) can be used to assess function of thecentromeres.

b. Telomeres

Preferred telomeres are the 1 kB synthetic telomere provided herein(see, Examples). A double synthetic telomere construct, which contains a1 kB synthetic telomere linked to a dominant selectable marker gene thatcontinues in an inverted orientation may be used for ease ofmanipulation. Such a double construct contains a series of TTAGGGrepeats 3′ of the marker gene and a series of repeats of the invertedsequence, i.e., GGGATT, 5′ of the marker gene as follows:(GGGATTT)_(n)- - -dominant marker gene- - -(TTAGGG)_(n). Using aninverted marker provides an easy means for insertion, such as by bluntend ligation, since only properly oriented fragments will be selected.

c. Megareplicator

The megareplicator sequences, such as the rDNA, provided herein arepreferred for use in in vitro constructs. The rDNA provides an origin ofreplication and also provides sequences that facilitate amplification ofthe artificial chromosome in vivo to increase the size of the chromosometo, for example accommodate increasing copies of a heterologous gene ofinterest as well as continuous high levels of expression of theheterologous genes.

d. Filler Heterochromatin

Filler heterochromatin, particularly satellite DNA, is included tomaintain structural integrity and stability of the artificial chromosomeand provide a structural base for carrying genes within the chromosome.The satellite DNA is typically A/T-rich DNA sequence, such as mousemajor satellite DNA, or G/C-rich DNA sequence, such as hamster naturalsatellite DNA. Sources of such DNA include any eukaryotic organisms thatcarry non-coding satellite DNA with sufficient A/T or G/C composition topromote ready separation by sequence, such as by FACS, or by densitygradients. The satellite DNA may also be synthesized by generatingsequence containing monotone, tandem repeats of highly A/T- or G/C-richDNA units.

The most suitable amount of filler heterochromatin for use inconstruction of the artificial chromosome may be empirically determinedby, for example, including segments of various lengths, increasing insize, in the construction process. Fragments that are too small to besuitable for use will not provide for a functional chromosome, which maybe evaluated in cell-based expression studies, or will result in achromosome of limited functional lifetime or mitotic and structuralstability.

e. Selectable Marker

Any convenient selectable marker may be used and at any convenient locusin the MAC.

2. Combination of the Isolated Chromosomal Elements

Once the isolated elements are obtained, they may be combined togenerate the complete, functional artificial chromosome. This assemblycan be accomplished for example, by In vitro ligation either insolution, LMP agarose or on microbeads. The ligation is conducted sothat one end of the centromere is directly joined to a telomere. Theother end of the centromere, which serves as the gene-carryingchromosome arm, is built up from a combination of satellite DNA and rDNAsequence and may also contain a selectable marker gene. Another telomereis joined to the end of the gene-carrying chromosome arm. Thegene-carrying arm is the site at which any heterologous genes ofinterest, for example, in expression of desired proteins encodedthereby, are incorporated either during in vitro construction of thechromosome or sometime thereafter.

3. Analysis and Testing of the Artificial Chromosome

Artificial chromosomes constructed in vitro may be tested forfunctionality in In vivo mammalian cell systems, using any of themethods described herein for the SATACs, minichromosomes, or known tothose of skill in the art.

4. Introduction of Desired Heterologous DNA into the in vitroSynthesized Chromosome

Heterologous DNA may be introduced into the in vitro synthesizedchromosome using routine methods of molecular biology, may be introducedusing the methods described herein for the SATACs, or may beincorporated into the in vitro synthesized chromosome as part of one ofthe synthetic elements, such as the heterochromatin. The heterologousDNA may be linked to a selected repeated fragment, and then theresulting construct may be amplified in vitro using the methods for suchin vitro amplification provided herein (see the Examples).

D. Introduction of Artificial Chromosomes into Cells, Tissues, Animalsand Plants

Suitable hosts for introduction of the MACs provided herein, include,but are not limited to, any animal or plant, cell or tissue thereof,including, but not limited to: mammals, birds, reptiles, amphibians,insects, fish, arachnids, tobacco, tomato, wheat, plants and algae. TheMACs, if contained in cells, may be introduced by cell fusion ormicrocell fusion or, if the MACs have been isolated from cells, they maybe introduced into host cells by any method known to those of skill inthis art, including but not limited to: direct DNA transfer,electroporation, lipid-mediated transfer, e.g., lipofection andliposomes, microprojectile bombardment, microinjection in cells andembryos, protoplast regeneration for plants, and any other suitablemethod [see, e.g., Weissbach et al. (1988) Methods for Plant MolecularBiology, Academic Press, N.Y., Section VIII, pp. 421-463; Grierson etal. (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9;see, also U.S. Pat. Nos. 5,491,075; 5,482,928; and 5,424,409; see, also,e.g., U.S. Pat. No. 5,470,708, which describes particle-mediatedtransformation of mammalian unattached cells].

Other methods for introducing DNA into cells include nuclearmicroinjection and bacterial protoplast fusion with intact cells.Polycations, such as polybrene and polyornithine, may also be used. Forvarious techniques for transforming mammalian cells, see e.g., Keown etal. Methods in Enzymology (1990) Vol. 185, pp. 527-537; and Mansour etal. (1988) Nature 336:348-352.

For example, isolated, purified artificial chromosomes can be injectedinto an embryonic cell line such as a human kidney primary embryoniccell line [ATCC accession number CRL 1573] or embryonic stem cells [see,e.g., Hogan et al. (1994) Manipulating the Mouse Embryo, A :LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,see, especially, pages 255-264 and Appendix 3].

Preferably the chromosomes are introduced by microinjection, using asystem such as the Eppendorf automated microinjection system, and grownunder selective conditions, such as in the presence of hygromycin B orneomycin.

1. Methods for Introduction of Chromosomes into Hosts

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. These methods include any,including those described herein, known to those of skill in the art.

a. DNA Uptake

For mammalian cells that do not have cell walls, the calcium phosphateprecipitation method for introduction of exogenous DNA [see, e.g.,Graham et al. (1978) Virology 52:456-457; Wigler et al. (1979) Proc.Natl. Acad. Sci. U.S.A. 76:1373-1376; and Current Protocols in MolecularBiology, Vol. 1, Wiley Inter-Science, Supplement 14, Unit 9.1.1-9.1.9(1990)] is often preferred. DNA uptake can be accomplished by DNA aloneor in the presence of polyethylene glycol [PEG-mediated gene transfer],which is a fusion agent, or by any variations of such methods known tothose of skill in the art [see, e.g., U.S. Pat. No. 4,684,611].

Lipid-mediated carrier systems are also among the preferred methods forintroduction of DNA into cells [see, e.g., Teifel et al. (1995)Biotechniques 19:79-80; Albrecht et al. (1996) Ann. Hematol. 72:73-79;Holmen et al. (1995) In Vitro Cell Dev. Biol. Anim. 31:347-351; Remy etal. (1994) Bioconjug. Chem. 5:647-654; Le Bolc'h et al. (1995)Tetrahedron Lett. 36:6681-6684; Loeffler et al. (1993) Meth. Enzymol.217:599-618]. Lipofection [see, e.g., Strauss (1996) Meth. Mol. Biol.54:307-327] may also be used to introduce DNA into cells. This method isparticularly well-suited for transfer of exogenous DNA into chickencells (e.g., chicken blastodermal cells and primary chicken fibroblasts;see Brazolot et al. (1991) Mol. Repro. Dev. 30:304-312). In particular,DNA of interest can be introduced into chickens in operative linkagewith promoters from genes, such as lysozyme and ovalbumin, that areexpressed in the egg, thereby permitting expression of the heterologousDNA in the egg.

Additional methods useful in the direct transfer of DNA into cellsinclude particle gun electrofusion [see, e.g., U.S. Pat. Nos. 4,955,378,4,923,814, 4,476,004, 4,906,576 and 4,441,972] and virion-mediated genetransfer.

A commonly used approach for gene transfer in land plants involves thedirect introduction of purified DNA into protoplasts. The three basicmethods for direct gene transfer into plant cells include: 1)polyethylene glycol [PEG]-mediated DNA uptake, 2)electroporation-mediated DNA uptake and 3) microinjection. In addition,plants may be transformed using ultrasound treatment [see, e.g.,International PCT application publication No. WO 91/00358].

b. Electroporation

Electroporation involves providing high-voltage electrical pulses to asolution containing a mixture of protoplasts and foreign DNA to createreversible pores in the membranes of plant protoplasts as well as othercells. Electroporation is generally used for prokaryotes or other cells,such as plants that contain substantial cell-wall barriers. Methods foreffecting electroporation are well known [see, e.g., U.S. Pat. Nos.4,784,737, 5,501,967, 5,501,662, 5,019,034, 5,503,999; see, also Frommetal. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:5824-5828].

For example, electroporation is often used for transformation of plants[see, e.g., Ag Biotechnology News 7:3 and 17 (September/October 1990)].In this technique, plant protoplasts are electroporated in the presenceof the DNA of interest that also includes a phenotypic marker.Electrical impulses of high field strength reversibly permeabilizebiomembranes allowing the introduction of the plasmids. Electroporatedplant protoplasts reform the cell wall, divide, and form plant callus.Transformed plant cells will be identified by virtue of the expressedphenotypic marker. The exogenous DNA may be added to the protoplasts inany form such as, for example, naked linear, circular or supercoiledDNA, DNA encapsulated in liposomes, DNA in spheroplasts, DNA in otherplant protoplasts, DNA complexed with salts, and other methods.

c. Microcells

The chromosomes can be transferred by preparing microcells containing anartificial chromosome and then fusing with selected target cells.Methods for such preparation and fusion of microcells are well known[see the Examples and also see, e.g., U.S. Pat. Nos. 5,240,840,4,806,476, 5,298,429, 5,396,767, Fournier (1981) Proc. Natl. Acad. Sci.U.S.A. 78:6349-6353; and Lambert et al. (1991) Proc. Natl. Acad. Sci.U.S.A. 88:5907-59]. Microcell fusion, using microcells that contain anartificial chromosome, is a particularly useful method for introductionof MACs into avian cells, such as DT40 chicken pre-B cells [for adescription of DT40 cell fusion, see, e.g., Dieken et al. (1996) NatureGenet. 12:174-182].

2. Hosts

Suitable hosts include any host known to be useful for introduction andexpression of heterologous DNA. Of particular interest herein, animaland plant cells and tissues, including, but not limited to insect cellsand larvae, plants, and animals, particularly transgenic (non-human)animals, and animal cells. Other hosts include, but are not limited tomammals, birds, particularly fowl such as chickens, reptiles,amphibians, insects, fish, arachnids, tobacco, tomato, wheat, monocots,dicots and algae, and any host into which introduction of heterologousDNA is desired. Such introduction can be effected using the MACsprovided herein, or, if necessary by using the MACs provided herein toidentify species-specific centromeres and/or functional chromosomalunits and then using the resulting centromeres or chromosomal units asartificial chromosomes, or alternatively, using the methods exemplifiedherein for production of MACs to produce species-specific artificialchromosomes.

a. Introduction of DNA into Embryos for Production of Transgenic(Non-Human) Animals and Introduction of DNA into Animal Cells

Transgenic (non-human) animals can be produced by introducing exogenousgenetic material into a pronucleus of a mammalian zygote bymicroinjection [see, e.g., U.S. Pat. Nos. 4,873,191 and 5,354,674; see,also, International PCT application publication No. WO 95/14769, whichis based on U.S. application Ser. No. 08/159,084]. The zygote is capableof development into a mammal. The embryo or zygote is transplanted intoa host female uterus and allowed to develop. Detailed protocols andexamples are set forth below.

Nuclear transfer [see, Wilmut et al. (1997) Nature 385:810-813,International PCT application Nos. WO 97/07669 and WO 97/076681. Brieflyin this method, the SATAC containing the genes of interest is introducedby any suitable method, into an appropriate donor cell, such as amammary gland cell, that contains totipotent nuclei. The diploid nucleusof the cell, which is either in G0 or G1 phase, is then introduced, suchas by cell fusion or microinjection, into an unactivated oöcyte,preferably enucleated cell, which is arrested in the metaphase of thesecond meiotic division. Enucleation may be effected by any suitablemethod, such as actual removal, or by treating with means, such asultraviolet light, that functionally remove the nucleus. The oöcyte isthen activated, preferably after a period of contact, about 6-20 hoursfor cattle, of the new nucleus with the cytoplasm, while maintainingcorrect ploidy, to produce a reconstituted embryo, which is thenintroduced into a host. Ploidy is maintained during activation, forexample, by incubating the reconstituted cell in the presence of amicrotubule inhibitor, such as nocodazole, colchicine, cocemid, andtaxol, whereby the DNA replicates once.

Transgenic chickens can be produced by injection of dispersedblastodermal cells from Stage X chicken embryos into recipient embryosat a similar stage of development [see e.g., Etches et al. (1993)Poultry Sci. 72:882-889; Petitte et al. (1990) Development 108:185-189].Heterologous DNA is first introduced into the donor blastodermal cellsusing methods such as, for example, lipofection [see, e.g., Brazolot etal. (1991) Mol. Repro. Dev. 30:304-312] or microcell fusion [see, e.g.,Dieken et al. (1996) Nature Genet. 12:174-182]. The transfected donorcells are then injected into recipient chicken embryos [see e.g.,Carsience et al. (1993) Development 117: 669-675]. The recipient chickenembryos within the shell are candled and allowed to hatch to yield agermline chimeric chicken.

DNA can be introduced into animal cells using any known procedure,including, but not limited to: direct uptake, incubation withpolyethylene glycol [PEG], microinjection, electroporation, lipofection,cell fusion, microcell fusion, particle bombardment, includingmicroprojectile bombardment [see, e.g., U.S. Pat. No. 5,470,708, whichprovides a method for transforming unattached mammalian cells viaparticle bombardment], and any other such method. For example, thetransfer of plasmid DNA in liposomes directly to human cells in situ hasbeen approved by the FDA for use in humans [see, e.g., Nabel, et al.(1990) Science 249:1285-1288 and U.S. Pat. No. 5,461,032].

b. Introduction of Heterologous DNA into Plants

Numerous methods for producing or developing transgenic plants areavailable to those of skill in the art. The method used is primarily afunction of the species of plant. These methods include, but are notlimited to: direct transfer of DNA by processes, such as PEG-induced DNAuptake, protoplast fusion, microinjection, electroporation, andmicroprojectile bombardment [see, e.g., Uchimiya et al. (1989) J. ofBiotech. 12: 1-20 for a review of such procedures, see, also, e.g., U.S.Pat. Nos. 5,436,392 and 5,489,520 and many others]. For purposes herein,when introducing a MAC, microinjection, protoplast fusion and particlegun bombardment are preferred.

Plant species, including tobacco, rice, maize, rye, soybean, Brassicanapus, cotton, lettuce, potato and tomato, have been used to producetransgenic plants. Tobacco and other species, such as petunias, oftenserve as experimental models in which the methods have been developedand the genes first introduced and expressed.

DNA uptake can be accomplished by DNA alone or in the presence of PEG,which is a fusion agent, with plant protoplasts or by any variations ofsuch methods known to those of skill in the art [see, e.g., U.S. Pat.No. 4,684,611 to Schilperoot et al.]. Electroporation, which involveshigh-voltage electrical pulses to a solution containing a mixture ofprotoplasts and foreign DNA to create reversible pores, has been used,for example, to successfully introduce foreign genes into rice andBrassica napus. Microinjection of DNA into plant cells, includingcultured cells and cells in intact plant organs and embryoids in tissueculture and microprojectile bombardment [acceleration of small highdensity particles, which contain the DNA, to high velocity with aparticle gun apparatus, which forces the particles to penetrate plantcell walls and membranes] have also been used. All plant cells intowhich DNA can be introduced and that can be regenerated from thetransformed cells can be used to produce transformed whole plants whichcontain the transferred artificial chromosome. The particular protocoland means for introduction of the DNA into the plant host may need to beadapted or refined to suit the particular plant species or cultivar.

C. Insect Cells

Insects are useful hosts for introduction of artificial chromosomes fornumerous reasons, including, but not limited to: (a) amplification ofgenes encoding useful proteins can be accomplished in the artificialchromosome to obtain higher protein yields in insect cells; (b) insectcells support required post-translational modifications, such asglycosylation and phosphorylation, that can be required for proteinbiological functioning; (c) insect cells do not support mammalianviruses, and, thus, eliminate the problem of cross-contamination ofproducts with such infectious agents; (d) this technology circumventstraditional recombinant baculovirus systems for production ofnutritional, industrial or medicinal proteins in insect cell systems;(e) the low temperature optimum for insect cell growth (28° C.) permitsreduced energy cost of production; (f) serum-free growth medium forinsect cells permits lower production costs; (g) artificialchromosome-containing cells can be stored indefinitely at lowtemperature; and (h) insect larvae will be biological factories forproduction of nutritional, medicinal or industrial proteins bymicroinjection of fertilized insect eggs [see, e.g., Joy et al. (1991)Current Science 66:145-150, which provides a method for microinjectingheterologous DNA into Bombyx mori eggs].

Either MACs or insect-specific artificial chromosomes [BUGACs] will beused to introduce genes into insects. As described in the Examples, itappears that MACs will function in insects to direct expression ofheterologous DNA contained thereon. For example, as described in theExamples, a MAC containing the B. mori actin gene promoter fused to thelacZ gene has been generated by transfection of EC3/7C5 cells with aplasmid containing the fusion gene. Subsequent fusion of the B. moricells with the transfected EC3/7C5 cells that survived selection yieldeda MAC-containing insect-mouse hybrid cell line in which β-galactosidaseexpression was detectable.

Insect host cells include, but are not limited to, hosts such asSpodoptera frugiperda [caterpillar], Aedes aegypti [mosquito], Aedesalbopictus [mosquito], Drosphila melanogaster [fruitfly], Bombyx mori[silkworm], Manduca sexta [tomato horn worm] and Trichoplusia ni[cabbage looper]. Efforts have been directed toward propagation ofinsect cells in culture. Such efforts have focused on the fall armyworm,Spodoptera frugiperda. Cell lines have been developed also from otherinsects such as the cabbage looper, Trichoplusia ni and the silkworm,Bombyx mori. It has also been suggested that analogous cell lines can becreated using the tomato hornworm, Manduca sexta. To introduce DNA intoan insect, it should be introduced into the larvae, and allowed toproliferate, and then the hemolymph recovered from the larvae so thatthe proteins can be isolated therefrom.

The preferred method herein for introduction of artificial chromosomesinto insect cells is microinjection [see, e.g., Tamura et al. (1991) BioInd. 8:26-31; Nikolaev et al. (1989) Mol. Biol. (Moscow) 23:1177-87; andmethods exemplified and discussed herein].

E. Applications for and Uses of Artificial Chromosomes

Artificial chromosomes provide convenient and useful vectors, and insome instances [e.g., in the case of very large heterologous genes] theonly vectors, for introduction of heterologous genes into hosts.Virtually any gene of interest is amenable to introduction into a hostvia artificial chromosomes. Such genes include, but are not limited to,genes that encode receptors, cytokines, enzymes, proteases, hormones,growth factors, antibodies, tumor suppressor genes, therapeutic productsand multigene pathways.

The artificial chromosomes provided herein will be used in methods ofprotein and gene product production, particularly using insects as hostcells for production of such products, and in cellular (e.g., mammaliancell) production systems in which the artificial chromomsomes(particularly MACs) provide a reliable, stable and efficient means foroptimizing the biomanufacturing of important compounds for medicine andindustry. They are also intended for use in methods of gene therapy, andfor production of transgenic plants and animals [discussed above, belowand in the EXAMPLES].

1. Gene Therapy

Any nucleic acid encoding a therapeutic gene product or product of amultigene pathway may be introduced into a host animal, such as a human,or into a target cell line for introduction into an animal, fortherapeutic purposes. Such therapeutic purposes include, genetic therapyto cure or to provide gene products that are missing or defective, todeliver agents, such as anti-tumor agents, to targeted cells or to ananimal, and to provide gene products that will confer resistance orreduce susceptibility to a pathogen or ameliorate symptoms of a diseaseor disorder. The following are some exemplary genes and gene products.Such exemplification is not intended to be limiting.

a. Anti-HIV Ribozymes

As exemplified below, DNA encoding anti-HIV ribozymes can be introducedand expressed in cells using MACs, including the euchromatin-basedminichromosomes and the SATACs. These MACs can be used to make atransgenic mouse that expresses a ribozyme and, thus, serves as a modelfor testing the activity of such ribozymes or from whichribozyme-producing cell lines can be made. Also, introduction of a MACthat encodes an anti-HIV ribozyme into human cells will serve astreatment for HIV infection. Such systems further demonstrate theviability of using any disease-specific ribozyme to treat or amelioratea particular disease.

b. Tumor Suppressor Genes

Tumor suppressor genes are genes that, in their wild-type alleles,express proteins that suppress abnormal cellular proliferation. When thegene coding for a tumor suppressor protein is mutated or deleted, theresulting mutant protein or the complete lack of tumor suppressorprotein expression may result in a failure to correctly regulatecellular proliferation. Consequently, abnormal cellular proliferationmay take place, particularly if there is already existing damage to thecellular regulatory mechanism. A number of well-studied human tumors andtumor cell lines have been shown to have missing or nonfunctional tumorsuppressor genes.

Examples of tumor suppression genes include, but are not limited to, theretinoblastoma susceptibility gene or RB gene, the p53 gene, the genethat is deleted in colon carcinoma [i.e., the DCC gene] and theneurofibromatosis type 1 [NF-1] tumor suppressor gene [see, e.g., U.S.Pat. No. 5,496,731; Weinberg et al. (1991) 254:1138-1146]. Loss offunction or inactivation of tumor suppressor genes may play a centralrole in the initiation and/or progression of a significant number ofhuman cancers.

The p53 Gene

Somatic cell mutations of the p53 gene are said to be the most frequentof the gene mutations associated with human cancer [see, e.g., Weinberget al. (1991) Science 254:1138-1146]. The normal or wild-type p53 geneis a negative regulator of cell growth, which, when damaged, favors celltransformation. The p53 expression product is found in the nucleus,where it may act in parallel or cooperatively with other gene products.Tumor cell lines in which p53 has been deleted have been successfullytreated with wild-type p53 vector to reduce tumorigenicity [see, Bakeret al. (1990) Science 249:912-915].

DNA encoding the p53 gene and plasmids containing this DNA are wellknown [see, e.g., U.S. Pat. No. 5,260,191; see, also Chen et al. (1990)Science 250:1576; Farrel et al. (1991) EMBO J. 10:2879-2887; plasmidscontaining the gene are available from the ATCC, and the sequence is inthe GenBank Database, accession nos. X54156, X60020, M14695, M16494,K03199].

c. The CFTR Gene

Cystic fibrosis [CF] is an autosomal recessive disease that affectsepithelia of the airways, sweat glands, pancreas, and other organs. Itis a lethal genetic disease associated with a defect in chloride iontransport, and is caused by mutations in the gene coding for the cysticfibrosis transmembrane conductance regulator [CFTR], a 1480 amino acidprotein that has been associated with the expression of chlorideconductance in a variety of eukaryotic cell types. Defects in CFTRdestroy or reduce the ability of epithelial cells in the airways, sweatglands, pancreas and other tissues to transport chloride ions inresponse to cAMP-mediated agonists and impair activation of apicalmembrane channels by cAMP-dependent protein kinase A [PKA]. Given thehigh incidence and devastating nature of this disease, development ofeffective CF treatments is imperative.

The CFTR gene [˜250 kb] can be transferred into a MAC for use, forexample, in gene therapy as follows. A CF-YAC [see Green et al. Science250:94-98] may be modified to include a selectable marker, such as agene encoding a protein that confers resistance to puromycin orhygromycin, and λ-DNA for use in site-specific integration into aneo-minichromosome or a SATAC. Such a modified CF-YAC can be introducedinto MAC-containing cells, such as EC3/7C5 or 19C5×Ha4 cells, by fusionwith yeast protoplasts harboring the modified CF-YAC or microinjectionof yeast nuclei harboring the modified CF-YAC into the cells. Stabletransformants are then selected on the basis of antibiotic resistance.These transformants will carry the modified CF-YAC within the MACcontained in the cells.

2. Animals, Birds, Fish and Plants that are Genetically Altered toPossess Desired Traits Such as Resistance to Disease

Artificial chromosomes are ideally suited for preparing animals,including vertebrates and invertebrates, including birds and fish aswell as mammals, that possess certain desired traits, such as, forexample, disease resistance, resistance to harsh environmentalconditions, altered growth patterns, and enhanced physicalcharacteristics.

One example of the use of artificial chromosomes in generatingdisease-resistant organisms involves the preparation of multivalentvaccines. Such vaccines include genes encoding multiple antigens thatcan be carried in a MAC, or species-specific artificial chromosome, andeither delivered to a host to induce immunity, or incorporated intoembryos to produce transgenic (non-human) animals and plants that areimmune or less susceptible to certain diseases.

Disease-resistant animals and plants may also be prepared in whichresistance or decreased susceptibility to disease is conferred byintroduction into the host organism or embryo of artificial chromosomescontaining DNA encoding gene products (e.g., ribozymes and proteins thatare toxic to certain pathogens) that destroy or attenuate pathogens orlimit access of pathogens to the host.

Animals and plants possessing desired traits that might, for example,enhance utility, processibility and commercial value of the organisms inareas such as the agricultural and ornamental plant industries may alsobe generated using artificial chromosomes in the same manner asdescribed above for production of disease-resistant animals and plants.In such instances, the artificial chromosomes that are introduced intothe organism or embryo contain DNA encoding gene products that serve toconfer the desired trait in the organism.

Birds, particularly fowl such as chickens, fish and crustaceans willserve as model hosts for production of genetically altered organismsusing artificial chromosomes.

3. Use of MACs and Other Artificial Chromosomes for Preparation andScreening of Libraries

Since large fragments of DNA can be incorporated into each artificialchromosome, the chromosomes are well-suited for use as cloning vehiclesthat can accommodate entire genomes in the preparation of genomic DNAlibraries, which then can be readily screened. For example, MACs may beused to prepare a genomic DNA library useful in the identification andisolation of functional centromeric DNA from different species oforganisms. In such applications, the MAC used to prepare a genomic DNAlibrary from a particular organism is one that is not functional incells of that organism. That is, the MAC does not stably replicate,segregate or provide for expression of genes contained within it incells of the organism. Preferably, the MACs contain an indicator gene(e.g., the lacZ gene encoding β-galactosidase or genes encoding productsthat confer resistance to antibiotics such as neomycin, puromycin,hygromycin) linked to a promoter that is capable of promotingtranscription of the indicator gene in cells of the organism. Fragmentsof genomic DNA from the organism are incorporated into the MACs, and theMACs are transferred to cells from the organism. Cells that contain MACsthat have incorporated functional centromeres contained within thegenomic DNA fragments are identified by detection of expression of themarker gene.

4. Use of MACs and Other Artificial Chromosomes for Stable, High-LevelProtein Production

Cells containing the MACs and/or other artificial chromosomes providedherein are advantageously used for production of proteins, particularlyseveral proteins from one cell line, such as multiple proteins involvedin a biochemical pathway or multivalent vaccines. The genes encoding theproteins are introduced into the artificial chromosomes which are thenintroduced into cells. Alternatively, the heterologous gene(s) ofinterest are transferred into a production cell line that alreadycontains artificial chromosomes in a manner that targets the gene(s) tothe artificial chromosomes. The cells are cultured under conditionswhereby the heterologous proteins are expressed. Because the proteinswill be expressed at high levels in a stable permanent extra-genomicchromosomal system, selective conditions are not required.

Any transfectable cells capable of serving as recombinant hostsadaptable to continuous propagation in a cell culture system [see, e.g.,McLean (1993) Trends In Biotech. 11:232-238] are suitable for use in anartificial chromosome-based protein production system. Exemplary hostcell lines include, but are not limited to, the following: Chinesehamster ovary (CHO) cells [see, e.g., Zang et al. (1995) Biotechnology13:389-392], HEK 293, Ltk⁻, COS-7, DG44, and BHK cells. CHO cells areparticularly preferred host cells. Selection of host cell lines for usein artificial chromosome-based protein production systems is within theskill of the art, but often will depend on a variety of factors,including the properties of the heterologous protein to be produced,potential toxicity of the protein in the host cell, any requirements forpost-translational modification (e.g., glycosylation, amination,phosphorylation) of the protein, transcription factors available in thecells, the type of promoter element(s) being used to drive expression ofthe heterologous gene, whether production will be completelyintracellular or the heterologous protein will preferably be secretedfrom the cell, and the types of processing enzymes in the cell.

The artificial chromosome-based system for heterologous proteinproduction has many advantageous features. For example, as describedabove, because the heterologous DNA is located in an independent,extra-genomic artificial chromosome (as opposed to randomly inserted inan unknown area of the host cell genome or located as extrachromosomalelement(s) providing only transient expression) it is stably maintainedin an active transcription unit and is not subject to ejection viarecombination or elimination during cell division. Accordingly, it isunnecessary to include a selection gene in the host cells and thusgrowth under selective conditions is also unnecessary. Furthermore,because the artificial chromosomes are capable of incorporating largesegments of DNA, multiple copies of the heterologous gene and linkedpromoter element(s) can be retained in the chromosomes, therebyproviding for high-level expression of the foreign protein(s).Alternatively, multiple copies of the gene can be linked to a singlepromoter element and several different genes may be linked in a fusedpolygene complex to a single promoter for expression of, for example,all the key proteins constituting a complete metabolic pathway [see,e.g., Beck von Bodman et al. (1995) Biotechnology 13:587-591].Alternatively, multiple copies of a single gene can be operativelylinked to a single promoter, or each or one or several copies may belinked to different promoters or multiple copies of the same promoter.Additionally, because artificial chromosomes have an almost unlimitedcapacity for integration and expression of foreign genes, they can beused not only for the expression of genes encoding end-products ofinterest, but also for the expression of genes associated with optimalmaintenance and metabolic management of the host cell, e.g., genesencoding growth factors, as well as genes that may facilitate rapidsynthesis of correct form of the desired heterologous protein product,e.g., genes encoding processing enzymes and transcription factors. TheMACS are suitable for expression of any proteins or peptides, includingproteins and peptides that require in vivo posttranslationalmodification for their biological activity. Such proteins include, butare not limited to antibody fragments, full-length antibodies, andmultimeric antibodies, tumor suppressor proteins, naturally occurring orartificial antibodies and enzymes, heat shock proteins, and others.

Thus, such cell-based “protein factories” employing MACs can generatedusing MACs constructed with multiple copies [theoretically an unlimitednumber or at least up to a number such that the resulting MAC is aboutup to the size of a genomic chromosome (i.e., endogenous)] ofprotein-encoding genes with appropriate promoters, or multiple genesdriven by a single promoter, i.e., a fused gene complex [such as acomplete metabolic pathway in plant expression system; see, e.g., Beckvon Bodman (1995) Biotechnology 13:587-591]. Once such MAC isconstructed, it can be transferred to a suitable cell culture system,such as a CHO cell line in protein-free culture medium [see, e.g.,(1995) Biotechnology 13:389-39] or other immortalized cell lines [see,e.g., (1993) TIBTECH 11:232-238] where continuous production can beestablished.

The ability of MACs to provide for high-level expression of heterologousproteins in host cells is demonstrated, for example, by analysis of theH1D3 and G3D5 cell lines described herein and deposited with the ECACC.Northern blot analysis of mRNA obtained from these cells reveals thatexpression of the hygromycin-resistance and β-galactosidase genes in thecells correlates with the amplicon number of the megachromosome(s)contained therein.

F. Methods for the Synthesis of DNA Sequences Containing Repeated DNAUnits

Generally, assembly of tandemly repeated DNA poses difficulties such asunambiguous annealing of the complementary oligos. For example,separately annealed products may ligate in an inverted orientation.Additionally, tandem or inverted repeats are particularly susceptible torecombination and deletion events that may disrupt the sequence.Selection of appropriate host organisms (e.g., rec⁻ strains) for use inthe cloning steps of the synthesis of sequences of tandemly repeated DNAunits may aid in reduction and elimination of such events.

Methods are provided herein for the synthesis of extended DNA sequencescontaining repeated DNA units. These methods are particularly applicableto the synthesis of arrays of tandemly repeated DNA units, which aregenerally difficult or not possible to construct utilizing other knowngene assembly strategies. A specific use of these methods is in thesynthesis of sequences of any length containing simple (e.g., rangingfrom 2-6 nucleotides) tandem repeats (such as telomeres and satelliteDNA repeats and trinucleotide repeats of possible clinical significance)as well as complex repeated DNA sequences. An particular example of thesynthesis of a telomere sequence containing over 150 successive repeatedhexamers utilizing these methods is provided herein.

The methods provided herein for synthesis of arrays of tandem DNArepeats are based in a series of extension steps in which successivedoublings of a sequence of repeats results in an exponential expansionof the array of tandem repeats. These methods provide several advantagesover previously known methods of gene assembly. For instance, thestarting oligonucleotides are used only once. The intermediates in, aswell as the final product of, the construction of the DNA arraysdescribed herein may be obtained in cloned form in a microbial organism(e.g., E. coli and yeast). Of particular significance, with regard tothese methods is the fact that sequence length increases exponentially,as opposed to linearly, in each extension step of the procedure eventhough only two oligonucleotides are required in the methods. Theconstruction process does not depend on the compatibility of restrictionenzyme recognition sequences and the sequence of the repeated DNAbecause restriction sites are used only temporarily during the assemblyprocedure. No adaptor is necessary, though a region of similar functionis located between two of the restriction sites employed in the process.The only limitation with respect to restriction site use is that the twosites employed in the method must not be present elsewhere in the vectorutilized in any cloning steps. These procedures can also be used toconstruct complex repeats with perfectly identical repeat units, such asthe variable number tandem repeat (VNTR) 3′ of the human apolipoproteinB100 gene (a repeat unit of 30 bp, 100% AT) or alphoid satellite DNA.

The method of synthesizing DNA sequences containing tandem repeats maygenerally be described as follows.

1. Starting Materials

Two oligonucleotides are utilized as starting materials. Oligonucleotide1 is of length k of repeated sequence (the flanks of which are notrelevant) and contains a relatively short stretch (60-90 nucleotides) ofthe repeated sequence, flanked with appropriately chosen restrictionsites: 5′-S1>>>>>>>>>>>>>>>>>>>>>>>>>>>S2_-3′wherein S1 is restriction site 1 cleaved by E1 [preferably an enzymeproducing a 3′-overhang PacI, PstI, SphI, NsiI, etc.) or blunt-end], S2is a second restriction site cleaved by E2 (preferably an enzymeproducing a 3′-overhang or one that cleaves outside the recognitionsequence, such as TspRI), >represents a simple repeat unit, and ‘______’denotes a short (8-10) nucleotide flanking sequence complementary tooligonucleotide 2:

-   -   3′______S3-5′        wherein S3 is a third restriction site for enzyme E3 and which        is present in the vector to be used during the construction.

Because there is a large variety of restriction enzymes that recognizemany different DNA sequences as cleavage sites, it should always bepossible to select sites and enzymes (preferably those that yield a3′-protruding end) suitable for these methods in connection with thesynthesis of any one particular repeat arrary. In most cases, only 1 (orperhaps 2) nucleotide(s) has of a restriction site is required to bepresent in the repeat sequence, and the remaining nucleotides of therestriction site can be removed, for example: PacI: TTAAT/TAA--(Klenow/dNTP) TAA-- PstI: CTGCA/G-- (Klenow/dNTP) G-- NsiI: ATGCA/T--(Klenow/dNTP) T-- KpnI: GGTAC/C-- (Klenow/dNTP) C--

Though there is no known restriction enzyme leaving a single A behind,this problem can be solved with enzymes leaving behind none at all, forexample: TaiI: ACGT/ (Klenow/dNTP) -- NIaIII: CATG/ (Klenow/dNTP) --

Additionally, if mung bean nuclease is used instead of Klenow, then thefollowing XbaI: T/CTAGA Mung bean nuclease A--

Furthermore, there are a number of restriction enzymes that cut outsideof the recognition sequence, and in this case, there is no limitation atall: TspRI NNCAGTGNN/-- (Klenow/dNTP) -- BsmI GAATG CN/-- (Klenow/dNTP)-- CTTAC/GN -- (Klenow/dNTP) --

2. Step 1—Annealing

Oligonucleotides 1 and 2 are annealed at a temperature selecteddepending on the length of overlap (typically in the range of 30-65 °C.).

3. Step 2—Generating a Double-Stranded Molecule

The annealed oligonucleotides are filled-in with Klenow polymerase inthe presence of dNTP to produce a double-stranded (ds) sequence:5′-S1>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>S2_S3-3′3′-S1<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<S2_S3-5′

4. Step 3—Incorporation of Double-Stranded DNA into a Vector

The double-stranded DNA is cleaved with restriction enzymes E1 and E3and subsequently ligated into a vector (e.g., pUC19 or a yeast vector)that has been cleaved with the same enzymes E1 and E3. The ligationproduct is used to transform competent host cells compatible with thevector being used (e.g., when pUC19 is used, bacterial cells such as E.coli DH5α are suitable hosts) which are then plated onto selectionplates. Recombinants can be identified either by color (e.g., by X-galstaining for β-galactosidase expression) or by colony hybridizationusing ³²P-labeled oligonucleotide 2 (detection by hybridization tooligonucleotide 2 is preferred because its sequence is removed in eachof the subsequent extension steps and thus is present only inrecombinants that contain DNA that has undergone successful extension ofthe repeated sequence).

5. Step 4—Isolation of Insert From the Plasmid

An aliquot of the recombinant plasmid containing k nucleotides of therepeat sequence is digested with restriction enzymes E1 and E3, and theinsert is isolated on a gel (native polyacrylamide while the insert isshort, but agarose can be used for isolation of longer inserts insubsequent steps). A second aliquot of the recombinant plasmid is cutwith enzymes E2 (treated with Klenow and dNTP to remove the 3′-overhang)and E3, and the large fragment (plasmid DNA plus the insert) isisolated.

6. Step 5—Extension of the DNA Sequence of k Repeats

The two DNAs (the S1-S3 insert fragment and the vector plus insert) areligated, plated to selective plates, and screened for extendedrecombinants as in Step 3. Now the length of the repeat sequence betweenrestriction sites is twice that of the repeat sequence in the previousstep, i.e., 2×k.

7. Step 6—Extension of the DNA Sequence of 2×k Repeats

Steps 4 and 5 are repeated as many times as needed to achieve thedesired repeat sequence size. In each extension cycle, the repeatsequence size doubles, i.e., if m is the number of extension cycles, thesize of the repeat sequence will be k×2^(m) nucleotides.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1

General Materials and Methods

The following materials and methods are exemplary of methods that areused in the following Examples and that can be used to prepare celllines containing artificial chromosomes. Other suitable materials andmethods known to those of skill in the art may used. Modifications ofthese materials and methods known to those of skill in the art may alsobe employed.

A. Culture of Cell Lines, Cell Fusion, and Transfection of Cells

1. Chinese hamster K-20 cells and mouse A9 fibroblast cells werecultured in F-12 medium. EC3/7 [see, U.S. Pat. No. 5,288,625, anddeposited at the European Collection of Animal cell Culture (ECACC)under accession no. 90051001; see, also Hadlaczky et al. (1991) Proc.Natl. Acad. Sci. U.S.A. 88:8106-8110 and U.S. application Ser. No.08/375,271] and EC3/7C5 [see, U.S. Pat. No. 5,288,625 and Praznovszky etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046] mouse celllines, and the KE1-2/4 hybrid cell line were maintained in F-12 mediumcontaining 400 μg/ml G418 [SIGMA, St. Louis, Mo.].

2. TF1004G19 and TF1004G-19C5 mouse cells, described below, and the19C5×Ha4 hybrid, described below, and its sublines were cultured in F-12medium containing up to 400 μg/ml Hygromycin B [Calbiochem]. LP11 cellswere maintained in F-12 medium containing 3-15 μg/ml Puromycin [SIGMA,St. Louis, Mo.].

3. Cotransfection of EC3/7C5 cells with plasmids [pH132, pCH110available from Pharmacia, see, also Hall et al. (1983) J. Mol. Appl.Gen. 2:101-109] and with λ DNA was conducted using the calcium phosphateDNA precipitation method [see, e.g., Chen et al. (1987) Mol. Cell. Biol.7:2745-2752], using 2-5 μg plasmid DNA and 20 μg λ phage DNA per 5×10⁶recipient cells.

4. Cell Fusion

Mouse and hamster cells were fused using polyethylene glycol [Davidsonet al. (1976) Som. Cell Genet. 2:165-176]. Hybrid cells were selected inHAT medium containing 400 μg/ml Hygromycin B.

Approximately 2×10⁷ recipient and 2×10⁶ donor cells were fused usingpolyethylene glycol [Davidson et al. (1976) Som. Cell Genet. 2:165-176].Hybrids were selected and maintained in F-12/HAT medium [Szybalsky etal. (1962) Natl. Cancer Inst. Monogr. 7:75-89] containing 10% FCS and400 μg/ml G418. The presence of “parental” chromosomes in the hybridcell lines was verified by in situ hybridization with species-specificprobes using biotin-labeled human and hamster genomic DNA, and a mouselong interspersed repetitive DNA [pMCPE1.51].

5. Microcell Fusion

Microcell-mediated transfer of artificial chromosomes from EC3/7C5 cellsto recipient cells was done according to Saxon et al. [(1985) Mol. Cell.Biol. 1:140-146] with the modifications of Goodfellow et al. [(1989)Techniques for mammalian genome transfer. In Genome Analysis a PracticalApproach. K. E. Davies, ed., IRL Press, Oxford, Washington D.C. pp.1-17]and Yamada et al. [(1990) Oncogene 5:1141-1147]. Briefly, 5×10⁶ EC3/7C5cells in a T25 flask were treated first with 0.05 μg/ml colcemid for 48hr and then with 10 μg/ml cytochalasin B for 30 min. The T25 flasks werecentrifuged on edge and the pelleted microcells were suspended in serumfree DME medium. The microcells were filtered through first a 5 micronand then a 3 micron polycarbonate filter, treated with 50 μg/ml ofphytohemagglutin, and used for polyethylene glycol mediated fusion withrecipient cells. Selection of cells containing the MMCneo was started 48hours after fusion in medium containing 400-800 μg/ml G418.

Microcells were also prepared from 1B3 and GHB42 donor cells as followsin order to be fused with E2D6K cells [a CHO K-20 cell line carrying thepuromycin N-acetyltransferase gene, i.e., the puromycin resistance gene,under the control of the SV40 early promoter]. The donor cells wereseeded to achieve 60-75% confluency within 24-36 hours. After that time,the cells were arrested in mitosis by exposure to colchicine (10 μg/ml)for 12 or 24 hours to induce micronucleation. To promote micronucleationof GHB42 cells, the cells were exposed to hypotonic treatment (10 min at37° C.). After colchicine treatment, or after colchicine and hypotonictreatment, the cells were grown in colchicine-free medium.

The donor cells were trypsinized and centrifuged and the pellets weresuspended in a 1:1 Percoll medium and incubated for 30-40 min at 37° C.After the incubation, 1-3×10⁷ cells (60-70% micronucleation index) wereloaded onto each Percoll gradient (each fusion was distributed on 1-2gradients). The gradients were centrifuged at 19,000 rpm for 80 min in aSorvall SS-34 rotor at 34-37° C. After centrifugation, two visible bandsof cells were removed, centrifuged at 2000 rpm, 10 min at 4° C.,resuspended and filtered through 8 μm pore size nucleopore filters.

The microcells prepared from the 1B3 and GHB42 cells were fused withE2D6K. The E2D6K cells were generated by CaPO₄ transfection of CHO K-20cells with pCHTV2. Plasmid pCHTV2 contains the puromycin-resistance genelinked to the SV40 promoter and polyadenylation signal, theSaccharomyces cerevisiae URA3 gene, 2.4- and 3.2-kb fragments of aChinese hamster chromosome 2-specific satellite DNA (HC-2 satellite; seeFatyol et al. (1994) Nuc. Acids Res. 22:3728-3736), two copies of thediptheria toxin-A chain gene (one linked to the herpes simplex virusthymidine kinase (HSV-TK) gene promoter and SV40 polyadenylation signaland the other linked to the HSV-TK promoter without a polyadenylationsignal), the ampicillin-resistance gene and the ColE1 origin ofreplication. Following transfection, puromycin-resistant colonies wereisolated. The presence of the pCHTV2 plasmid in the E2D6K cell line wasconfirmed by nucleic acid amplification of DNA isolated from the cells.

The purified microcells were centrifuged as described above andresuspended in 2 ml of phytohemagglutinin-P (PHA-P, 100 μg/ml). Themicrocell suspension was then added to a 60-70% confluent recipientculture of E2D6K cells. The preparation was incubated at roomtemperature for 30-40 min to agglutinate the microcells. After the PHA-Pwas removed, the cells were incubated with 1 ml of 50%polyethylene-glycol (PEG) for one min. The PEG was removed and theculture was washed three times with F-12 medium without serum. The cellswere incubated in non-selective medium for 48-60 hours. After this time,the cell culture was trypsinized and plated in F-12 medium containing400 μg/ml hygromycin B and 10 g/ml puromycin to select against theparental cell lines.

Hybrid clones were isolated from the cells that had been cultured inselective medium. These clones were then analyzed for expression ofβ-galactosidase by the X-gal staining method. Four of five hybrid clonesanalyzed that had been generated by fusion of GHB42 microcells withE2D6K cells yielded positive staining results indicating expression ofβ-galactosidase from the lacZ gene contained in the megachromosomecontributed by the GHB42 cells. Similarly, a hybrid clone that had beengenerated by fusion of 1B3 microcells with E2D6K cells yielded positivestaining results indicating expression of β-galactosidase from the lacZgene contained in the megachromosome contributed by the 1B3 cells. Insitu hybridization analysis of the hybrid clones is also performed toanalyze the mouse chromosome content of the mouse-hamster hybrid cells.

B. Chromosome Banding

Trypsin G-banding of chromosomes was performed using the method of Wang& Fedoroff [(1972) Nature 235:52-54], and the detection of constitutiveheterochromatin with the BSG. C-banding method was done according toSumner [(1972) Exp. Cell Res. 75:304-306]. For the detection ofchromosome replication by bromodeoxyuridine [BrdU] incorporation, theFluorescein Plus Giemsa [FPG] staining method of Perry & Wolff [(1974)Nature 251:156-158] was used.

C. Immunolabelling of Chromosomes and in situ Hybridization

Indirect immunofluorescence labelling with human anti-centromere serumLU851 [Hadlaczky et al. (1986) Exp. Cell Res. 167:1-15], and indirectimmunofluorescence and in situ hybridization on the same preparationwere performed as described previously [see, Hadlaczky et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110, see, also U.S. applicationSer. No. 08/375,271]. Immunolabelling with fluorescein-conjugatedanti-BrdU monoclonal antibody [Boehringer] was performed according tothe procedure recommended by the manufacturer, except that for treatmentof mouse A9 chromosomes, 2 M hydrochloric acid was used at 37° C. for 25min, and for chromosomes of hybrid cells, 1 M hydrochloric acid was usedat 37° C. for 30 min.

D. Scanning Electron Microscopy

Preparation of mitotic chromosomes for scanning electron microscopyusing osmium impregnation was performed as described previously [Sumner(1991) Chromosoma 100:410-418]. The chromosomes were observed with aHitachi S-800 field emission scanning electron microscope operated withan accelerating voltage of 25 kV.

E. DNA Manipulations, Plasmids and Probes

1. General Methods

All general DNA manipulations were performed by standard procedures[see, e.g., Sambrook et al. (1989) Molecular cloning: A LaboratoryManual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.].The mouse major satellite probe was provided by Dr. J. B. Rattner[University of Calgary, Alberta, Canada]. Cloned mouse satellite DNAprobes [see Wong et al. (1988) Nucl. Acids Res. 16:11645-11661],including the mouse major satellite probe, were gifts from Dr. J. B.Rattner, University of Calgary. Hamster chromosome painting was donewith total hamster genomic DNA, and a cloned repetitive sequencespecific to the centromeric region of chromosome 2 [Fátyol et al. (1994)Nucl. Acids Res. 22:3728-3736] was also used. Mouse chromosome paintingwas done with a cloned long interspersed repetitive sequence [pMCP1.51]specific for the mouse euchromatin.

For cotransfection and for in situ hybridization, the pCH 110β-galactosidase construct [Pharmacia or Invitrogen], and λcl 875 Sam7phage DNA [New England Biolabs] were used.

2. Construction of Plasmid pPuroTel

Plasmid pPuroTel, which carries a Puromycin-resistance gene and a cloned2.5 kb human telomeric sequence [see SEQ ID No. 3], was constructed fromthe pBabe-puro retroviral vector [Morgenstern et al. (1990) Nucl. AcidsRes. 18:3587-3596; provided by Dr. L. Székely (Microbiology andTumorbiology Center, Karolinska Institutet, Stockholm); see, alsoTonghua et al. (1995) Chin. Med. J. (Beijing, Engl. Ed.) 108:653-659;Couto et al. (1994) Infect. Immun. 62:2375-2378; Dunckley et al. (1992)FEBS Lett. 296:128-34; French et al. (1995) Anal. Biochem. 228:354-355;Liu et al. (1995) Blood 85:1095-1103; International PCT application Nos.WO 9520044; WO 9500178, and WO 9419456].

F. Deposited Cell Lines

Cell lines KE1-2/4, EC3/7C5, TF1004G19C5, 19C5×Ha4, G3D5 and H1D3 havebeen deposited in accord with the Budapest Treaty at the EuropeanCollection of Animal Cell Culture (ECACC) under Accession Nos. 96040924,96040925, 96040926, 96040927, 96040928 and 96040929, respectively. Thecell lines were deposited on Apr. 9, 1996, at the European Collection ofAnimal Cell Cultures (ECACC) Vaccine Research and Production Laboratory,Public Health Laboratory Service, Centre for Appliced Microbiology andResearch, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom. Thedeposits were made in the name of Gyula Hadlaczky of H. 6723, SZEGED,SZAMOS U.1.A. IX. 36. HUNGARY, who has authorized reference to thedeposited cell lines in this application.

EXAMPLE 2

Preparation of EC3/7, EC3/7C5 and Related Cell Lines The EC3/7 cell lineis an LMTK⁻ mouse cell line that contains the neo-centromere. TheEC3/7C5 cell line is a single-cell subclone of EC3/7 that contains theneo-minichromosome.

A. EC3/7 Cell Line

As described in U.S. Pat. No. 5,288,625 [see, also Praznovszky et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046 and Hadlaczky et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110] de novo centromereformation occurs in a transformed mouse LMTK⁻ fibro-blast cell line[EC3/7] after cointegration of λ constructs [λCM8 and λgtWESneo]carrying human and bacterial DNA.

By cotransfection of a 14 kb human DNA fragment cloned in λ [λCM8] and adominant marker gene [λgtWESneo], a selectable centromere linked to adominant marker gene [neo-centromere] was formed in mouse LMTK⁻ cellline EC3/7 [Hadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:8106-8110, see FIG. 1]. Integration of the heterologous DNA [the λDNA and marker gene-encoding DNA] occurred into the short arm of anacrocentric chromosome [chromosome 7 (see, FIG. 1B)], where anamplification process resulted in the formation of the new centromere[neo-centromere (see FIG. 1C)]. On the dicentric chromosome (FIG. 1C),the newly formed centromere region contains all the heterologous DNA(human, λ, and bacterial) introduced into the cell and an activecentromere.

Having two functionally active centromeres on the same chromosome causesregular breakages between the centromeres [see, FIG. 1E]. The distancebetween the two centromeres on the dicentric chromosome is estimated tobe ˜10-15 Mb, and the breakage that separates the minichromosomeoccurred between the two centromeres. Such specific chromosome breakagesresult in the appearance [in approximately 10% of the cells] of achromosome fragment that carries the neo-centromere [FIG. 1F]. Thischromosome fragment is principally composed of human, λ, plasmid, andneomycin-resistance gene DNA, but it also has some mouse chromosomalDNA. Cytological evidence suggests that during the stabilization of theMMCneo, there was an inverted duplication of the chromosome fragmentbearing the neo-centromere. The size of minichromosomes in cell linescontaining the MMCneo is approximately 20-30 Mb; this finding indicatesa two-fold increase in size.

From the EC3/7 cell line, which contains the dicentric chromosome [FIG.1E], two sublines [EC3/7C5 and EC3/7C6] were selected by repeatedsingle-cell cloning. In these cell lines, the neo-centromere was foundexclusively on a small chromosome [neo-minichromosome], while theformerly dicentric chromosome carried detectable amounts of theexogenously-derived DNA sequences but not an active neo-centromere[FIGS. 1F and 1G].

The minichromosomes of cell lines EC3/7C5 and EC3/7C6 are similar. Nodifferences are detected in their architectures at either thecytological or molecular level. The minichromosomes wereindistinguishable by conventional restriction endonuclease mapping or bylong-range mapping using pulsed field electrophoresis and Southernhybridization. The cytoskeleton of cells of the EC3/7C6 line showed anincreased sensitivity to colchicine, so the EC3/7C5 line was used forfurther detailed analysis.

B. Preparation of the EC3/7C5 and EC3/7C6 Cell Lines

The EC3/7C5 cells, which contain the neo-minichromosome, were producedby subcloning the EC3/7 cell line in high concentrations of G418[40-fold the lethal dose] for 350 generations. Two single cell-derivedstable cell lines [EC3/7C5 and EC3/7C6] were established. These celllines carry the neo-centromere on minichromosomes and also contain theremaining fragment of the dicentric chromosome. Indirectimmunofluorescence with anti-centromere antibodies and subsequent insitu hybridization experiments demonstrated that the minichromosomesderived from the dicentric chromosome. In EC3/7C5 and EC3/7C6 cell lines(140 and 128 metaphases, respectively) no intact dicentric chromosomeswere found, and minichromosomes were detected in 97.2% and 98.1% of thecells, respectively. The minichromosomes have been maintained for over150 cell generations. They do contain the remaining portion of theformerly dicentric chromosome.

Multiple copies of telomeric DNA sequences were detected in the markercentromeric region of the remaining portion of the formerly dicentricchromosome by in situ hybridization. This indicates that mouse telomericsequences were coamplified with the foreign DNA sequences. These stableminichromosome-carrying cell lines provide direct evidence that theextra centromere is functioning and is capable of maintaining theminichromosomes [see, U.S. Pat. No. 5,288,625].

The chromosome breakage in the EC3/7 cells, which separates theneo-centromere from the mouse chromosome, occurred in the G-bandpositive “foreign” DNA region. This is supported by the observation oftraces of λ and human DNA sequences at the broken end of the formerlydicentric chromosome. Comparing the G-band pattern of the chromosomefragment carrying the neo-centromere with that of the stableneo-minichromosome, reveals that the neo-minichromosome is an invertedduplicate of the chromosome fragment that bears the neo-centromere. Thisis also evidenced by the observation that although theneo-minichromosome carries only one functional centromere, both ends ofthe minichromosome are heterochromatic, and mouse satellite DNAsequences were found in these heterochromatic regions by in situhybridization.

These two cell lines, EC3/7C5 and EC3/7C6, thus carry a selectablemammalian minichromosome [MMCneo] with a centromere linked to a dominantmarker gene [Hadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:8106-8110]. MMCneo is intended to be used as a vector forminichromosome-mediated gene transfer and has been used as model of aminichromosome-based vector system.

Long range mapping studies of the MMCneo indicated that human DNA andthe neomycin-resistance gene constructs integrated into the mousechromosome separately, followed by the amplification of the chromosomeregion that contains the exogenous DNA. The MMCneo contains about 30-50copies of the λCM8 and λgtWESneo DNA in the form of approximately 160 kbrepeated blocks, which together cover at least a 3.5 Mb region. Inaddition to these, there are mouse telomeric sequences [Praznovszky etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046] and any DNA ofmouse origin necessary for the correct higher-ordered structuralorganization of chromatids.

Using a chromosome painting probe mCPE1.51 [mouse long interspersedrepeated DNA], which recognizes exclusively euchromatic mouse DNA,detectable amounts of interspersed repeat sequences were found on theMMCneo by in situ hybridization. The neo-centromere is associated with asmall but detectable amount of satellite DNA. The chromosome breakagethat separates the neo-centromere from the mouse chromosome occurs inthe “foreign” DNA region. This is demonstrated by the presence of λ andhuman DNA at the broken end of the formerly dicentric chromosome. Atboth ends of the MMCneo, however, there are traces of mouse majorsatellite DNA as evidenced by in situ hybridization. This observationsuggests that the doubling in size of the chromosome fragment carryingthe neo-centromere during the stabilization of the MMCneo is a result ofan inverted duplication. Although mouse telomere sequences, whichcoamplified with the exogenous DNA sequences during the neo-centromereformation, may provide sufficient telomeres for the MMCneo, theduplication could have supplied the functional telomeres for theminichromosome.

The nucleotide sequence of portions of the neo-minichromosomes wasdetermined as follows. Total DNA was isolated from EC3/7C5 cellsaccording to standard procedures. The DNA was subjected to nucleic acidamplification using the Expand Long Template PCR system [BoehringerMannheim] according to the manufacturer's procedures. The amplificationprocedure required only a single 33-mer oligonucleotide primercorresponding to sequence in a region of the phage λ right arm, which iscontained in the neo-minichromosome. The sequence of thisoligonucleotide is set forth as the first 33 nucleotides of SEQ ID No.13. Because the neo-minichromosome contains a series of inverted repeatsof this sequence, the single oligonucleotide was used as a forward andreverse primer resulting in amplification of DNA positioned between setsof inverted repeats of the phage λ DNA. Three products were obtainedfrom the single amplification reaction, which suggests that the sequenceof the DNA located between different sets of inverted repeats maydiffer. In a repeating nucleic acid unit within an artificialchromosome, minor differences may be present and may occur duringculturing of cells containing the artificial chromosome. For example,base pair changes may occur as well as integration of mobile geneticelements and deletions of repeated sequences.

Each of the three products was subjected to DNA sequence analysis. Thesequences of the three products are set forth in SEQ ID Nos. 13, 14, and15, respectively. To be certain that the sequenced products wereamplified from the neo-minichromosome, control amplifications wereconducted using the same primers on DNA isolated from negative controlcell lines (mouse Ltk⁻ cells) lacking minichromosomes and the formerlydicentric chromosome, and positive control cell lines [the mouse-hamsterhybrid cell line GB43 generated by treating 19C5×Ha4 cells (see FIG. 4)with BrdU followed by growth in G418-containing selective medium andretreatment with BrdU] containing the neo-minichromosome only. Only thepositive control cell line yielded the three amplification products; noamplification product was detected in the negative control reaction. Theresults obtained in the positive control amplification also demonstratethat the neo-minichromosome DNA, and not the fragment of the formerlydicentric mouse chromosome, was amplified.

The sequences of the three amplification products were compared to thosecontained in the Genbank/EMBL database. SEQ ID Nos. 13 and 14 showedhigh (˜96%) homology to portions of DNA from intracisternal A-particlesfrom mouse. SEQ ID No. 15 showed no significant homology with sequencesavailable in the database. All three of these sequences may be used forgenerating gene targeting vectors as homologous DNAs to theneo-minichromosome.

C. Isolation and Partial Purification of Minichromosomes

Mitotic chromosomes of EC3/7C5 cells were isolated as described byHadlaczky et al. [(1981) Chromosoma 81:537-555], using aglycine-hexylene glycol buffer system [Hadlaczky et al. (1982)Chromosoma 86:643-659]. Chromosome suspensions were centrifuged at1,200×g for 30 minutes. The supernatant containing minichromosomes wascentrifuged at 5,000×g for 30 minutes and the pellet was resuspended inthe appropriate buffer. Partially purified minichromosomes were storedin 50% glycerol at −20° C.

D. Stability of the MMCneo Maintenance and neo Expression

EC3/7C5 cells grown in non-selective medium for 284 days and thentransferred to selective medium containing 400 μg/ml G418 showed a 96%plating efficiency (colony formation) compared to control cells culturedpermanently in the presence of G418. Cytogenetic analysis indicated thatthe MMCneo is stably maintained at one copy per cell under selective andnon-selective culture conditions. Only two metaphases with two MMCneowere found in 2,270 metaphases analyzed.

Southern hybridization analysis showed no detectable changes in DNArestriction patterns, and similar hybridization intensities wereobserved with a neo probe when DNA from cells grown under selective ornon-selective culture conditions were compared.

Northern analysis of RNA transcripts from the neo gene isolated fromcells grown under selective and non-selective conditions showed onlyminor and not significant differences. Expression of the neo genepersisted in EC3/7C5 cells maintained in F-12 medium free of G418 for290 days under non-selective culture conditions. The long-termexpression of the neo gene(s) from the minichromosome may be influencedby the nuclear location of the MMCneo. In situ hybridization experimentsrevealed a preferential peripheral location of the MMCneo in theinterphase nucleus. In more than 60% of the 2,500 nuclei analyses, theminichromosome was observed at the perimeter of the nucleus near thenuclear envelope.

EXAMPLE 3

Minichromosome Transfer and Production of the λ-neo-chromosome

A. Minichromosome Transfer

The neo-minichromosome [referred to as MMCneo, FIG. 2C] has been usedfor gene transfer by fusion of minichromosome-containing cells [EC3/7C5or EC3/7C6] with different mammalian cells, including hamster and human.Thirty-seven stable hybrid cell lines have been produced. Allestablished hybrid cell lines proved to be true hybrids as evidenced byin situ hybridization using biotinylated human, and hamster genomic, orpMCPE1.51 mouse long interspersed repeated DNA probes for “chromosomepainting”. The MMCneo has also been successfully transferred into mouseA9, L929 and pluripotent F9 teratocarcinoma cells by fusion ofmicrocells derived from EC3/7C5 cells. Transfer was confirmed by PCR,Southern blotting and in situ hybridization with minichromosome-specificprobes. The cytogenetic analysis confirmed that, as expected formicrocell fusion, a few cells [1-5%] received [or retained] the MMCneo.

These results demonstrate that the MMCneo is tolerated by a wide rangeof cells. The prokaryotic genes and the extra dosage for the human and λsequences carried on the minichromosome seem to be not disadvantageousfor tissue culture cells.

The MMCneo is the smallest chromosome of the EC3/7C5 genome and isestimated to be approximately 20-30 Mb, which is significantly smallerthan the majority of the host cell (mouse) chromosomes. By virtue of thesmaller size, minichromosomes can be partially purified from asuspension of isolated chromosomes by a simple differentialcentrifugation. In this way, minichromosome suspensions of 15-20% purityhave been prepared. These enriched minichromosome preparations can beused to introduce, such as by microinjection or lipofection, theminichromosome into selected target cells. Target cells includetherapeutic cells that can be use in methods of gene therapy, and alsoembryonic cells for the preparation of transgenic (non-human) animals.

The MMCneo is capable of autonomous replication, is stably maintained incells, and permits persistent expression of the neo gene(s), even afterlong-term culturing under non-selective conditions. It is anon-integrative vector that appears to occupy a territory near thenuclear envelope. Its peripheral localization in the nucleus may have animportant role in maintaining the functional integrity and stability ofthe MMCneo. Functional compartmentalization of the host nucleus may havean effect on the function of foreign sequences. In addition, MMCneocontains megabases of λ DNA sequences that should serve as a target sitefor homologous recombination and thus integration of desired gene(s)into the MMCneo. It can be transferred by cell and microcell fusion,microinjection, electroporation, lipid-mediated carrier systems orchromosome uptake. The neo-centromere of the MMCneo is capable ofmaintaining and supporting the normal segregation of a larger 150-200 Mbλneo-chromosome. This result demonstrates that the MMCneo chromosomeshould be useful for carrying large fragments of heterologous DNA.

B. Production of the λneo-chromosome

In the hybrid cell line KE1-2/4 made by fusion of EC3/7 and Chinesehamster ovary cells [FIG. 2], the separation of the neo-centromere fromthe dicentric chromosome was associated with a further amplificationprocess. This amplification resulted in the formation of a stablechromosome of average size [i.e., the λneo-chromosome; see, Praznovszkyet al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046]. Theλneo-chromosome carries a terminally located functional centromere andis composed of seven large amplicons containing multiple copies of λ,human, bacterial, and mouse DNA sequences [see FIG. 2]. The ampliconsare separated by mouse major satellite DNA [Praznovszky et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88:11042-11046] which forms narrow bandsof constitutive heterochromatin between the amplicons.

EXAMPLE 4

Formation of the “Sausage Chromosome” [SC]

The findings set forth in the above EXAMPLES demonstrate that thecentromeric region of the mouse chromosome 7 has the capacity forlarge-scale amplification [other results indicate that this capacity isnot unique to chromosome 7]. This conclusion is further supported byresults from cotransfection experiments, in which a second dominantselectable marker gene and a non-selected marker gene were introducedinto EC3/7C5 cells carrying the formerly dicentric chromosome 7 and theneo-minichromosome. The EC3/7C5 cell line was transformed with λ phageDNA, a hygromycin-resistance gene construct [pH132], and β-galactosidasegene construct [pCH110]. Stable transformants were selected in thepresence of high concentrations [400 μg/ml] Hygromycin B, and analyzedby Southern hybridization. Established transformant cell lines showingmultiple copies of integrated exogenous DNA were studied by in situhybridization to localize the integration site(s), and by LacZ stainingto detect β-galactosidase expression.

A. Materials and Methods

1. Construction of pH132

The pH132 plasmid carries the hygromycin B resistance gene and theanti-HIV-1 gag ribozyme [see, SEQ ID NO. 6 for DNA sequence thatcorresponds to the sequence of the ribozyme] under control of theβ-actin promoter. This plasmid was constructed from pHyg plasmid [Sugdenet al. (1985) Mol. Cell. Biol. 5:410-413; a gift from Dr. A. D. Riggs,Beckman Research Institute, Duarte; see, also, e.g., U.S. Pat. No.4,997,7641, and from pPC-RAG12 plasmid [see, Chang et al. (1990) ClinBiotech 2:23-31; provided by Dr. J. J. Rossi, Beckman ResearchInstitute, Duarte; see, also U.S. Pat. Nos. 5,272,262, 5,149,796 and5,144,019, which describes the anti-HIV gag ribozyme and construction ofa mammalian expression vector containing the ribozyme insert linked tothe β-actin promoter and SV40 late gene transcriptional termination andpolyA signals]. Construction of pPC-RAG12 involved insertion of theribozyme insert flanked by BamHI linkers was into BamHI-digestedpHβ-Apr-1gpt [see, Gunning et al. (1987) Proc. Natl. Acad. Sci. U.S.A.84:4831-4835, see, also U.S. Pat. No. 5,144,019].

Plasmid pH132 was constructed as follows. First, pPC-RAG12 [described byChang et al. (1990) Clin. Biotech. 2:23-31] was digested with BamHI toexcise a fragment containing an anti-HIV ribozyme gene [referred to asribozyme D by Chang et al. [(1990) Clin. Biotech. 2:23-31]; see alsoU.S. Pat. No. 5,144,019 to Rossi et al., particularly FIG. 4 of thepatent] flanked by the human β-actin promoter at the 5′ end of the geneand the SV40 late transcriptional termination and polyadenylationsignals at the 3′ end of the gene. As described by Chang et al. [(1990)Clin. Biotech. 2:23-31], ribozyme D is targeted for cleavage of thetranslational initiation region of the HIV gag gene. This fragment ofpPC-RAG12 was subcloned into pBluescript-KS(+) [Stratagene, La Jolla,Calif.] to produce plasmid 132. Plasmid 132 was then digested with XhoIand EcoRI to yield a fragment containing the ribozyme D gene flanked bythe β-actin promoter at the 5′ end and the SV40 termination andpolyadenylation signals at the 3′ end of the gene. This fragment wasligated to the largest fragment generated by digestion of pHyg [Sugdenet al. (1985) Mol. Cell. Biol. 5:410-413] with EcoRI and SalI to yieldpH132. Thus, pH132 is an ˜9.3 kb plasmid containing the followingelements: the β-actin promoter linked to an anti-HIV ribozyme genefollowed by the SV40 termination and polyadenylation signals, thethymidine kinase gene promoter linked to the hygromycin-resistance genefollowed by the thymidine kinase gene polyadenylation signal, and the E.coli ColE1 origin of replication and the ampicillin-resistance gene.

The plasmid pHyg [see, e.g., U.S. Pat. Nos. 4,997,764, 4,686,186 and5,162,215], which confers resistance to hygromycin B usingtranscriptional controls from the HSV-1 tk gene, was originallyconstructed from pKan2 [Yates et al. (1984) Proc. Natl. Acad. Sci.U.S.A. 81:3806-3810] and pLG89 [see, Gritz et al. (1983) Gene25:179-188]. Briefly pKan2 was digested with SmaI and BqlII to removethe sequences derived from transposon Tn5. The hygromycin-resistance hphgene was inserted into the digested pKan2 using blunt-end ligation atthe SnaI site and “sticky-end” ligation [using 1 Weiss unit of T4 DNAligase (BRL) in 20 microliter volume] at the BglII site. The SmaI andBglII sites of pKan2 were lost during ligation.

The resulting plasmid pH132, produced from introduction of the anti-HIVribozyme construct with promoter and polyA site into pHyg, includes theanti-HIV ribozyme under control of the β-actin promoter as well as thehygromycin-resistance gene under control of the TK promoter.

2. Chromosome Banding

Trypsin G-banding of chromosomes was performed as described in EXAMPLE1.

3. Cell Cultures

TF1004G19 and TF1004G-19C5 mouse cells and the 19C5×Ha4 hybrid,described below, and its sublines were cultured in F-12 mediumcontaining 400 μg/ml Hygromycin B [Calbiochem].

B. Cotransfection of EC3/7C5 to Produce TF1004G19

Cotransfection of EC3/7C5 cells with plasmids [pH132, pCH110 availablefrom Pharmacia, see, also Hall et al. (1983) J. Mol. Appl. Gen.2:101-109] and with λ DNA [λcl 875 Sam 7(New England Biolabs)] wasconducted using the calcium phosphate DNA precipitation method [see,e.g., Chen et al. (1987) Mol. Cell. Biol. 7:2745-2752], using 2-5 μgplasmid DNA and 20 μg λ phage DNA per 5×10⁶ recipient cells.

C. Cell Lines Containing the Sausage Chromosome

Analysis of one of the transformants, designated TF1004G19, revealedthat it has a high copy number of integrated pH132 and pCH110 sequences,and a high level of β-galactosidase expression. G-banding and in situhybridization with a human probe [CM8; see, e.g., U.S. application Ser.No. 08/375,271] revealed unexpectedly that integration had occurred inthe formerly dicentric chromosome 7 of the EC3/7C5 cell line.Furthermore, this chromosome carried a newly formed heterochromaticchromosome arm. The size of this heterochromatic arm varied between ˜150and ˜800 Mb in individual metaphases.

By single cell cloning from the TF1004G19 cell line, a subcloneTF1004G-19C5 [FIG. 2D], which carries a stable chromosome 7 with a˜100-150 Mb heterochromatic arm [the sausage chromosome] was obtained.This cell line has been deposited in the ECACC under Accession No.96040926. This chromosome arm is composed of four to five satellitesegments rich in satellite DNA, and evenly spaced integratedheterologous “foreign” DNA sequences. At the end of the compactheterochromatic arm of the sausage chromosome, a less condensedeuchromatic terminal segment is regularly observed. This subclone wasused for further analyses.

D. Demonstration that the Sausage Chromosome is Derived from theFormerly Dicentric Chromosome

In situ hybridization with I phage and pH132 DNA on the TF1004G-19C5cell line showed positive hybridization only on the minichromosome andon the heterochromatic arm of the “sausage” chromosome [FIG. 2D]. Itappears that the “sausage” chromosome [herein also referred to as theSC] developed from the formerly dicentric chromosome (FD) of the EC3/7C5cell line.

To establish this, the integration sites of pCH110 and pH132 plasmidswere determined. This was accomplished by in situ hybridization on thesecells with biotin-labeled subfragments of the hygromycin-resistance geneand the β-galactosidase gene. Both experiments resulted in narrowhybridizing bands on the heterochromatic arm of the sausage chromosome.The same hybridization pattern was detected on the sausage chromosomeusing a mixture of biotin-labeled λ probe and pH132 plasmid, proving thecointegration of λ phages, pH132 and pCH110 plasmids.

To examine this further, the cells were cultured in the presence of theDNA-binding dye Hoechst 33258. Culturing of mouse cells in the presenceof this dye results in under-condensation of the pericentricheterochromatin of metaphase chromosomes, thereby permitting betterobservation of the hybridization pattern. Using this technique, theheterochromatic arm of the sausage chromosome of TF1004G-19C5 cellsshowed regular under-condensation revealing the details of the structureof the “sausage” chromosome by in situ hybridization. Results of in situhybridization on Hoechst-treated TF1004G-19C5 cells with biotin-labeledsubfragments of hygromycin-resistance and β-galactosidase genes showsthat these genes are localized only in the heterochromatic arm of thesausage chromosome. In addition, an equal banding hybridization patternwas observed. This pattern of repeating units [amplicons] clearlyindicates that the sausage chromosome was formed by an amplificationprocess and that the λ phage, pH132 and pCH110 plasmid DNA sequencesborder the amplicons.

In another series of experiments using fluorescence in situhybridization [FISH] carried out with mouse major satellite DNA, themain component of the mouse pericentric heterochromatin, the resultsconfirmed that the amplicons of the sausage chromosome are primarilycomposed of satellite DNA.

E. The Sausage Chromosome Has One Centromere

To determine whether mouse centromeric sequences had participated in theamplification process forming the “sausage” chromosome and whether ornot the amplicons carry inactive centromeres, in situ hybridization wascarried out with mouse minor satellite DNA. Mouse minor satellite DNA islocalized specifically near the centromeres of all mouse chromosomes.Positive hybridization was detected in all mouse centromeres includingthe sausage chromosome, which, however, only showed a positive signal atthe beginning of the heterochromatic arm.

Indirect immunofluorescence with a human anti-centromere anti-body [LU851] which recognizes only functional centromeres [see, e.g., Hadlaczkyet a[. (1989) Chromosoma 97:282-288] proved that the sausage chromosomehas only one active centromere. The centromere comes from the formerlydicentric part of the chromosome and co-localizes with the in situhybridization signal of the mouse minor DNA probe.

F. The Selected and Non-Selected Heterologous DNA in the Heterochromatinof the Sausage Chromosome is Expressed

1. High Levels of the Heterologous Genes are Expressed

The TF1004G-19C5 cell line thus carries multiple copies ofhygromycin-resistance and β-galactosidase genes localized only in theheterochromatic arm of the sausage chromosome. The TF1004G-19C5 cellscan grow very well in the presence of 200 μg/ml or even 400 μg/mlhygromycin B. [The level of expression was determined by Northernhybridization with a subfragment of the hygromycin-resistance gene andsingle copy gene.]

The expression of the non-selected β-galactosidase gene in theTF1004G-19C5 transformant was detected with LacZ staining of the cells.By this method one hundred percent of the cells stained dark blue,showing that there is a high level of β-galactosidase expression in allof TF1004G-19C5 cells.

2. The Heterologous Genes that are Expressed are in the Heterochromatinof the Sausage Chromosome

To demonstrate that the genes localized in the constitutiveheterochromatin of the sausage chromosome provide the hygromycinresistance and the LacZ staining capability of TF1004G-19C5transformants [i.e. β-gal expression], PEG-induced cell fusion betweenTF1004G-19C5 mouse cells and Chinese hamster ovary cells was performed.The hybrids were selected and maintained in HAT medium containing G418[400 μg/ml and hygromycin [200 μg/ml]. Two hybrid clones designated19C5×Ha3 and 19C5×Ha4, which have been deposited in the ECACC underAccession No. 96040927, were selected. Both carry the sausage chromosomeand the minichromosome.

Twenty-seven single cell derived colonies of the 19C5×Ha4 hybrid weremaintained and analyzed as individual subclones. In situ hybridizationwith hamster and mouse chromosome painting probes and hamster chromosome2-specific probes verified that the 19C5×Ha4 clone contains the completeChinese hamster genome and a partial mouse genome. All 19C5×Ha4subclones retained the hamster genome, but different subclones showeddifferent numbers of mouse chromosomes indicating the preferentialelimination of mouse chromosomes.

To promote further elimination of mouse chromosomes, hybrid cells wererepeatedly treated with BrdU. The BrdU treatments, which destabilize thegenome, result in significant loss of mouse chromosomes. TheBrdU-treated 19C5×Ha4 hybrid cells were divided to three groups. Onegroup of the hybrid cells (GH) were maintained in the presence ofhygromycin (200 μg/ml) and G418 (400 μg/ml), and the other two groups ofthe cells were cultured under G418 (G) or hygromycin (H) selectionconditions to promote the elimination of the sausage chromosome orminichromosome.

One month later, single cell derived subclones were established fromthese three subcultures of the 19C5×Ha4 hybrid line. The subclones weremonitored by in situ hybridization with biotin-labeled λ phage andhamster chromosome painting probes. Four individual clones [G2B5, G3C5,G4D6, G2B4] selected in the presence of G418 that had lost the sausagechromosome but retained the minichromosome were found. Under hygromycinselection only one subclone [H1D3] lost the minichromosome. In thisclone the megachromosome [see Example 5] was present.

Since hygromycin-resistance and β-galactosidase genes were thought to beexpressed from the sausage chromosome, the expression of these genes wasanalyzed in the four subclones that had lost the sausage chromosome. Inthe presence of 200 μg/ml hygromycin, one hundred percent of the cellsof four individual subclones died. In order to detect theβ-galactosidase expression hybrid, subclones were analyzed by LacZstaining. One hundred percent of the cells of the four subclones thatlost the sausage chromosome also lost the LacZ staining capability. Allof the other hybrid subclones that had not lost the sausage chromosomeunder the non-selective culture conditions showed positive LacZstaining.

These findings demonstrate that the expression of hygromycin-resistanceand β-galactosidase genes is linked to the presence of the sausagechromosome. Results of in situ hybridizations show that the heterologousDNA is expressed from the constitutive heterochromatin of the sausagechromosome.

In situ hybridization studies of three other hybrid subclones [G2C6,G2D1, and G4D5] did not detect the presence of the sausage chromosome.By the LacZ staining method, some stained cells were detected in thesehybrid lines, and when these subclones were transferred to hygromycinselection some colonies survived. Cytological analysis and in situhybridization of these hygromycin-resistant colonies revealed thepresence of the sausage chromosome, suggesting that only the cells ofG2C6, G2D1 and G4D5 hybrids that had not lost the sausage chromosomewere able to preserve the hygromycin resistance and β-galactosidaseexpression. These results confirmed that the expression of these genesis linked to the presence of the sausage chromosome. The level ofβ-galactosidase expression was determined by the immunoblot techniqueusing a monoclonal antibody.

Hygromycin resistance and β-galactosidase expression of the cells whichcontained the sausage chromosome were provided by the genes localized inthe mouse pericentric heterochromatin. This was demonstrated byperforming Southern DNA hybridizations on the hybrid cells that lack thesausage chromosome using PCR-amplified subfragments ofhygromycin-resistance and β-galactosidase genes as probes. None of thesubclones showed hybridization with these probes; however, all of theanalyzed clones contained the minichromosome. Other hybrid clones thatcontain the sausage chromosome showed intense hybridization with theseDNA probes. These results lead to the conclusion that hygromycinresistance and β-galactosidase expression of the cells that contain thesausage chromosome were provided by the genes localized in the mousepericentric heterochromatin.

EXAMPLE 5

The Gigachromosome

As described in Example 4, the sausage chromosome was transferred intoChinese hamster cells by cell fusion. Using Hygromycin B/HAT and G418selection, two hybrid clones 19C5×Ha3 and 19C5×Ha4 were produced thatcarry the sausage chromosome. In situ hybridization, using hamster andmouse chromosome-painting probes and a hamster chromosome 2-specificprobe, verified that clone 19C5×Ha4 contains a complete Chinese hamstergenome as well as partial mouse genomes. Twenty-seven separate coloniesof 19C5×Ha4 cells were maintained and analyzed as individual subclones.Twenty-six out of 27 subclones contained a morphologically unchangedsausage chromosome.

In one subclone of the 19C5×Ha3 cell line, 19C5×Ha47 [see FIG. 2E], theheterochromatic arm of the sausage chromosome became unstable and showedcontinuous intrachromosomal growth. In extreme cases, the amplifiedchromosome arm exceeded 1000 Mb in size (gigachromosome).

EXAMPLE 6

The Stable Megachromosome

A. Generation of Cell Lines Containing the Megachromosome

All 19C5×Ha4 subclones retained a complete hamster genome, but differentsubclones showed different numbers of mouse chromosomes, indicating thepreferential elimination of mouse chromosomes. As described in Example4, to promote further elimination of mouse chromosomes, hybrid cellswere treated with BrdU, cultured under G418 (G) or hygromycin (H)selection conditions followed by repeated treatment with 10⁻⁴ M BrdU for16 hours and single cell subclones were established. The BrdU treatmentsappeared to destabilize the genome, resulting in a change in the sausagechromosome as well. A gradual increase in a cell population in which afurther amplification had occurred was observed. In addition to the˜100-150 Mb heterochromatic arm of the sausage chromosome, an extracentromere and a ˜150-250 Mb heterochromatic chromosome arm were formed,which differed from those of mouse chromosome 7. By the acquisition ofanother euchromatic terminal segment, a new submetacentric chromosome(megachromosome) was formed. Seventy-nine individual subclones wereestablished from these BrdU-treated cultures by single-cell cloning: 42subclones carried the intact megachromosome, 5 subclones carried thesausage chromosome, and in 32 subclones fragments or translocatedsegments of the megachromosome were observed. Twenty-six subclones thatcarried the megachromosome were cultured under non-selective conditionsover a two-month period. In 19 out of 26 subclones, the megachromosomewas retained. Those subclones which lost the megachromosomes all becamesensitive to Hygromycin B and had no β-galactosidase expression,indicating that both markers were linked to the megachromosome.

Two sublines (G3D5 and H1D3), which were chosen for further experiments,showed no changes in the morphology of the megachromosome during morethan 100 generations under selective conditions. The G3D5 cells had beenobtained by growth of 19C5×Ha4 cells in G418-containing medium followedby repeated BrdU treatment, whereas H1D3 cells had been obtained byculturing 19C5×Ha4 cells in hygromycin-containing medium followed byrepeated BrdU treatment.

B. Structure of the Megachromosome

The following results demonstrate that, apart from the euchromaticterminal segments, the integrated foreign DNA (and as in the exemplifiedembodiments, rDNA sequence), the whole megachromosome is constitutiveheterochromatin, containing a tandem array of at least 40 [˜7.5 Mb]blocks of mouse major satellite DNA [see FIGS. 2 and 3]. Four satelliteDNA blocks are organized into a giant palindrome [amplicon] carryingintegrated exogenous DNA sequences at each end. The long and short armsof the submetacentric megachromosome contains 6 and 4 amplicons,respectively. It is of course understood that the specific organizationand size of each component can vary among species, and also thechromosome in which the amplification event initiates.

1. The Megachromosome is Composed Primarily of Heterochromatin

Except for the terminal regions and the integrated foreign DNA, themegachromosome is composed primarily of heterochromatin. This wasdemonstrated by C-banding of the megachromosome, which resulted inpositive staining characteristic of constitutive heterochromatin. Apartfrom the terminal regions and the integrated foreign DNA, the wholemegachromosome appears to be heterochromatic. Mouse major satellite DNAis the main component of the pericentric, constitutive heterochromatinof mouse chromosomes and represents ˜10% of the total DNA [Waring et al.(1966) Science 154:791-794]. Using a mouse major satellite DNA probe forin situ hybridization, strong hybridization was observed throughout themegachromosome, except for its terminal regions. The hybridizationshowed a segmented pattern: four large blocks appeared on the short armand usually 4-7 blocks were seen on the long arm. By comparing thesesegments with the pericentric regions of normal mouse chromosomes thatcarry ˜15 Mb of major satellite DNA, the size of the blocks of majorsatellite DNA on the megachromosome was estimated to be ˜30 Mb.

Using a mouse probe specific to euchromatin [pMCPE1.51; a mouse longinterspersed repeated DNA probe], positive hybridization was detectedonly on the terminal segments of the megachromosome of the H1D3 hybridsubline. In the G3D5 hybrids, hybridization with a hamster-specificprobe revealed that several megachromosomes contained terminal segmentsof hamster origin on the long arm. This observation indicated that theacquisition of the terminal segments on these chromosomes happened inthe hybrid cells, and that the long arm of the megachromosome was therecently formed one arm. When a mouse minor satellite probe was used,specific to the centromeres of mouse chromosomes [Wong et al. (1988)Nucl. Acids Res. 16:11645-11661], a strong hybridization signal wasdetected only at the primary constriction of the megachromosome, whichcolocalized with the positive immuno-fluorescence signal produced withhuman anti-centromere serum [LU851].

In situ hybridization experiments with pH132, pCH110, and λ DNA probesrevealed that all heterologous DNA was located in the gaps between themouse major satellite DNA segments. Each segment of mouse majorsatellite DNA was bordered by a narrow band of integrated heterologousDNA, except at the second segment of the long arm where a double band ofheterologous DNA existed, indicating that the major satellite DNAsegment was missing or considerably reduced in size here. Thischromosome region served as a useful cytological marker in identifyingthe long arm of the megachromosome. At a frequency of 10⁻⁴,“restoration” of these missing satellite DNA blocks was observed in onechromatid, when the formation of a whole segment on one chromatidoccurred.

After Hoechst 33258 treatment (50 μg/ml for 16 hours), themegachromosome showed undercondensation throughout its length except forthe terminal segments. This made it possible to study the architectureof the megachromosome at higher resolution. In situ hybridization withthe mouse major satellite probe on undercondensed megachromosomesdemonstrated that the ˜30 Mb major satellite segments were composed offour blocks of ˜7.5 Mb separated from each other by a narrow band ofnon-hybridizing sequences [FIG. 3]. Similar segmentation can be observedin the large block of pericentric heterochromatin in metacentric mousechromosomes from the LMTK⁻ and A9 cell lines.

2. The Megachromosome is Composed of Segments Containing Two Tandem ˜7.5Mb Blocks Followed by Two Inverted Blocks

Because of the asymmetry in thymidine content between the two strands ofthe DNA of the mouse major satellite, when mouse cells are grown in thepresence of BrdU for a single S phase, the constitutive heterochromatinshows lateral asymmetry after FPG staining. Also, in the 19C5×Ha4hybrids, the thymidine-kinase [Tk] deficiency of the mouse fibroblastcells was complemented by the hamster Tk gene, permitting BrdUincorporation experiments.

A striking structural regularity in the megachromosome was detectedusing the FPG technique. In both chromatids, alternating dark and lightstaining that produced a checkered appearance of the megachromosome wasobserved. A similar picture was obtained by labelling withfluorescein-conjugated anti-BrdU antibody. Comparing these pictures tothe segmented appearance of the megachromosome showed that one dark andone light FPG band corresponded to one ˜30 Mb segment of themegachromosome. These results suggest that the two halves of the ˜30 Mbsegment have an inverted orientation. This was verified by combining insitu hybridization and immunolabelling of the incorporated BrdU withfluorescein-conjugated anti-BrdU antibody on the same chromosome. Sincethe ˜30 Mb segments [or amplicons] of the megachromosome are composed offour blocks of mouse major satellite DNA, it can be concluded that twotandem ˜7.5 Mb blocks are followed by two inverted blocks within onesegment.

Large-scale mapping of megachromosome DNA by pulsed-fieldelectrophoresis and Southern hybridization with “foreign” DNA probesrevealed a simple pattern of restriction fragments. Using endonucleaseswith none, or only a single cleavage site in the integrated foreign DNAsequences, followed by hybridization with a hyg probe, 1-4 predominantfragments were detected. Since the megachromosome contains 10-12amplicons with an estimated 3-8 copies of hyg sequences per amplicon(30-90 copies per megachromosome), the small number of hybridizingfragments indicates the homogeneity of DNA in the amplified segments.

3. Scanning Electron Microscopy of the Megachromosome Confirmed theAbove Findings

The homogeneous architecture of the heterochromatic arms of themegachromosome was confirmed by high resolution scanning electronmicroscopy. Extended arms of megachromosomes, and the pericentricheterochromatic region of mouse chromosomes, treated with Hoechst 33258,showed similar structure. The constitutive heterochromatic regionsappeared more compact than the euchromatic segments. Apart from theterminal regions, both arms of the megachromosome were completelyextended, and showed faint grooves, which should correspond to theborder of the satellite DNA blocks in the non-amplified chromosomes andin the megachromosome. Without Hoechst treatment, the grooves seemed tocorrespond to the amplicon borders on the megachromosome arms. Inaddition, centromeres showed a more compact, finely fibrous appearancethan the surrounding heterochromatin.

4. The Megachromosome of 1B3 Cells Contains rRNA Gene Sequence

The sequence of the megachromosome in the region of the sites ofintegration of the heterologous DNA was investigated by isolation ofthese regions through using cloning methods and sequence analysis of theresulting clones. The results of this analysis revealed that theheterologous DNA was located near mouse ribosomal RNA gene (i.e., rDNA)sequences contained in the megachromosome.

a. Cloning of Regions of the Megachromosomes in Which Heterologous DNAhad Integrated

Megachromosomes were isolated from 1B3 cells (which were generated byrepeated BrdU treatment and single cell cloning of H1×HE41 cells (seeFIG. 4) and which contain a truncated megachromosome) usingfluorescence-activated cell sorting methods as described herein (seeExample 10). Following separation of the SATACs (megachromosomes) fromthe endogenous chromosomes, the isolated megachromosomes were stored inGH buffer (100 mM glycine, 1% hexylene glycol, pH 8.4-8.6 adjusted withsaturated calcium hydroxide solution; see Example 10) and centrifugedinto an agarose bed in 0.5 M EDTA.

Large-scale mapping of the megachromosome around the area of the site ofintegration of the heterologous DNA revealed that it is enriched insequence containing rare-cutting enzyme sites, such as the recognitionsite for NotI. Additionally, mouse major satellite DNA (which makes upthe majority of the megachromosome) does not contain NotI recognitionsites. Therefore, to facilitate isolation of regions of themegachromosome associated with the site of integration of theheterologous DNA, the isolated megachromosomes were cleaved with NotI, arare cutting restriction endonuclease with an 8-bp GC recognition site.Fragments of the megachromosome were inserted into plasmid pWE15(Stratagene, La Jolla, Calif.) as follows. Half of a 100-μl low meltingpoint agarose block (mega-plug) containing the isolated SATACs wasdigested with NotI overnight at 37° C. Plasmid pWE15 was similarlydigested with NotI overnight. The mega-plug was then melted and mixedwith the digested plasmid, ligation buffer and T4 ligase. Ligation wasconducted at 16° C. overnight. Bacterial DH5α cells were transformedwith the ligation product and transformed cells were plated onto LB/Ampplates. Fifteen to twenty colonies were grown on each plate for a totalof 189 colonies. Plasmid DNA was isolated from colonies that survivedgrowth on LB/Amp medium and was analyzed by Southern blot hybridizationfor the presence of DNA that hybridized to a pUC19 probe. This screeningmethodology assured that all clones, even clones lacking an insert butyet containing the pWE15 plasmid, would be detected. Any clonescontaining insert DNA would be expected to contain containnon-satellite, GC-rich megachromosome DNA sequences located at the siteof integration of the heterologous DNA. All colonies were positive forhybridizing DNA.

Liquid cultures of all 189 transformants were used to generate cosmidminipreps for analysis of restriction sites within the insert DNA. Sixof the original 189 cosmid clones conatained an insert. These cloneswere designated as follows: 28 (˜9-kb insert), 30 (˜9-kb insert), 60(˜4-kb insert), 113 (˜9-kb insert), 157 (˜9-kb insert) and 161 (˜9-kbinsert). Restriction enzyme analysis indicated that three of the clones(113, 157 and 16 1) contained the same insert.

b. In situ Hybridization Experiments Using Isolated Segments of theMegachromosome as Probes

Insert DNA from clones 30, 113, 157 and 161 was purified, labeled andused as probes in in situ hybridization studies of several cell lines.Counterstaining of the cells with propidium iodide facilitatedidentification of the cytological sites of the hybridization signals.The locations of the signals detected within the cells are summarized inthe following table: CELL TYPE PROBE LOCATION OF SIGNAL Human LymphocyteNo. 161 4-5 pairs of acrocentic chromosomes (male) at centromericregions. Mouse Spleen No. 161 Acrocentric ends of 4 pairs ofchromosomes. EC3/7C5 Cells No. 161 Minichromosome and the end of theformerly dicentric chromosome. Pericentric heterochromatin of one of themetacentric mouse chromosomes. Centromeric region of some of the othermouse chromosomes. K20 No. 30 Ends of at least 6 pairs of ChineseHamster chromosomes. An interstitial signal Cells on a short chromosome.HB31 Cells No. 30 Acrocentric ends of at least 12 pairs (mouse-hamsterhybrid of chromosomes. Centromeres of cells derived from H1D3 certainchromosomes and the cells by repeated BrdU megachromosome. Borders ofthe treatment and single amplicons of the megachromosome. cell cloningwhich carries the megachromosome) Mouse Spleen Cells No. 30 Similar tosignal observed for probe no. 161. Centromeres of 5 pairs ofchromosomes. Weak cross- hybridization to pericentric heterochromatin.HB31 Cells No. 113 Similar to signal observed for probe no. 30. MouseSpleen Cells No. 113 Centromeric region of 5 pairs of chromosomes. K20Cells No. 113 At least 6 pairs of chromosomes. Weak signal at sometelomeres and several interspersed signals. Human Lymphocyte No. 157Similar to signal observed for probe Cells (male) no. 161.

c. Southern Blot Hybridization Using Isolated Segments of theMegachromosome as Probes

DNA was isolated from mouse spleen tissue, mouse LMTK⁻ cells, K20Chinese hamster ovary cells, EJ30 human fibroblast cells and H1D3 cells.The isolated DNA and lambda phage DNA, was subjected to Southern blothybridization using inserts isolated from megachromosome clone nos. 30,113, 157 and 161 as probes. Plasmid pWE15 was used as a negative controlprobe. Each of the four megachromosome clone inserts hybridized in amulti-copy manner (as demonstrated by the intensity of hybridization andthe number of hybridizing bands) to all of the DNA samples, except thelambda phage DNA. Plasmid pWE15 hybridized to lambda DNA only.

d. Sequence Analysis of Megachromosome Clone No. 161

Megachromosome clone no. 161 appeared to show the strongesthybridization in the in situ and Southern hybridization experiments andwas chosen for analysis of the insert sequence. The sequence analysiswas approached by first subcloning the insert of cosmid clone no. 161 toobtain five subclones as follows.

To obtain the end fragments of the insert of clone no. 161, the clonewas digested with NotI and BamHI and ligated with NotI/BamHI-digestedpBluescript KS (Stratagene, La Jolla, Calif.). Two fragments of theinsert of clone no. 161 were obtained: a 0.2-kb and a 0.7-kb insertfragment. To subclone the internal fragment of the insert of clone no.161, the same digest was ligated with BamHI-digested pUC19. Threefragments of the insert of clone no. 161 were obtained: a 0.6-kb, a1.8-kb and a 4.8-kb insert fragment.

The ends of all the subcloned insert fragments were first sequencedmanually. However, due to their extremely high GC content,autoradiographs were difficult to interpret and sequencing was repeatedusing an ABI sequencer and the dye-terminator cycle protocol. Acomparison of the sequence data to sequences in the GENBANK databaserevealed that the insert of clone no. 161 corresponds to an internalsection of the mouse ribosomal RNA gene (rDNA) repeat unit betweenpositions 7551-15670 as set forth in GENBANK accession no. X82564, whichis provided as SEQ ID NO. 16 herein. The sequence data obtained for theinsert of clone no. 161 is set forth in SEQ ID NOS. 18-24. Specifically,the individual subclones corresponded to the following positions inGENBANK accession no. X82564 (i.e., SEQ ID NO. 16) and in SEQ ID NOs.18-24: Start End Subclone in X82564 Site SEQ ID No. 161k1 7579 7755NotI, BamHI 18 161m5 7756 8494 BamHI 19 161m7 8495 10231 BamHI 20 (showsonly sequence corresponding to nt. 8495-8950), 21 (shows only sequencecorresponding to nt. 9851-10231) 161m12 10232 15000 BamHI 22 (shows onlysequence corresponding to nt. 10232-10600), 23 (shows only sequencecorresponding to nt. 14267-15000), 161k2 15001 15676 NotI, BamHI 24

The sequence set forth in SEQ ID NOs. 18-24 diverges in some positionsfrom the sequence presented in positions 7551-15670 of GENBANK accessionno. X82564. Such divergence may be attributable to random mutationsbetween repeat units of rDNA. The results of the sequence analysis ofclone no. 161, which reveal that it corresponds to rDNA, correlate withthe appearance of the in situ hybridization signal it generated in humanlymphocytes and mouse spleen cells. The hybridization signal was clearlyobserved on acrocentric chromosomes in these cells, and such types ofchromosomes are known to include rDNA adjacent to the pericentricsatellite DNA on the short arm of the chromosome. Furthermore, rRNAgenes are highly conserved in mammals as supported by the cross-specieshybridization of clone no. 161 to human chromosomal DNA.

To isolate amplification-replication control regions such as those foundin rDNA, it may be possible to subject DNA isolated frommegachromosome-containing cells, such as H1D3 cells, to nucleic acidamplification using, e.g., the polymerase chain reaction (PCR) with thefollowing primers: amplification control element forward primer (1-30)(SEQ ID NO. 25) 5′-GAGGAATTCCCCATCCCTAATCCAGATTGGTG-3′ amplificationcontrol element reverse primer (2142-2111) (SEQ ID NO. 26)5′-AAACTGCAGGCCGAGCCACCTCTCTTCTGTGTTTG-3′ origin of replication regionforward primer (2116-2141) (SEQ ID NO. 27)5′-AGGAATTCACAGAAGAGAGGTGGCTCGGCCTGC-3′ origin of replication regionreverse primer (5546-5521) (SEQ ID NO. 28)5′-AGCCTGCAGGAAGTCATACCTGGGGAGGTGGCCC-3′C. Summary of the Formation of the Megachromosome

FIG. 2 schematically sets forth events leading to the formation of astable megachromosome beginning with the generation of a dicentricchromosome in a mouse LMTK⁻ cell line: (A) A single E-type amplificationin the centromeric region of the mouse chromosome 7 followingtransfection of LMTK⁻ cells with λCM8 and λgtWESneo generates theneo-centromere linked to the integrated foreign DNA, and forms adicentric chromosome. Multiple E-type amplification forms theλneo-chromosome, which was derived from chromosome 7 and stabilized in amouse-hamster hybrid cell line; (B) Specific breakage between thecentromeres of a dicentric chromosome 7 generates a chromosome fragmentwith the neo-centromere, and a chromosome 7 with traces of foreign DNAat the end; (C) Inverted duplication of the fragment bearing theneo-centromere results in the formation of a stable neo-minichromosome;(D) Integration of exogenous DNA into the foreign DNA region of theformerly dicentric chromosome 7 initiates H-type amplification, and theformation of a heterochromatic arm. By capturing a euchromatic terminalsegment, this new chromosome arm is stabilized in the form of the“sausage” chromosome; (E) BrdU treatment and/or drug selection appearsto induce further H-type amplification, which results in the formationof an unstable gigachromosome: (F) Repeated BrdU treatments and/or drugselection induce further H-type amplification including a centromereduplication, which leads to the formation of another heterochromaticchromosome arm. It is split off from the chromosome 7 by chromosomebreakage and acquires a terminal segment to form the stablemegachromosome.

D. Expression of β-galactosidase and hygromycin Transferase Genes inCell Lines Carrying the Megachromosome or Derivatives Thereof

The level of heterologous gene (i.e., β-galactosidase and hygromycintransferase genes) expression in cell lines containing themegachromosome or a derivative thereof was quantitatively measured. Therelationship between the copy-number of the heterologous genes and thelevel of protein expressed therefrom was also determined.

1. Materials and Methods

a. Cell Lines

Heterologous gene expression levels of H1D3 cells, carrying a 250-400 Mbmegachromosome as decribed above, and mM2C1 cells, carrying a 50-60 Mbmicro-megachromosome, were quantitatively evaluated. mM2C1 cells weregenerated by repeated BrdU treatment and single cell cloning of theH1×He41 cell line (mouse-hamster-human hybrid cell line carrying themegachromosome and a single human chromosome with CD4 and neo^(r) genes;see FIG. 4). The cell lines were grown under standard conditions in F12medium under selective (120 μg/ml hygromycin) or non-selectiveconditions.

b. Preparation of cell Extract for β-galactosidase Assays

Monolayers of mM2C1 or H1D3 cell cultures were washed three times withphosphate-buffered saline (PBS). Cells were scraped by rubber policemenand suspended and washed again in PBS. Washed cells were resuspendedinto 0.25 M Tris-HCl, pH 7.8, and disrupted by three cycles of freezingin liquid nitrogen and thawing at 37° C. The extract was clarified bycentrifugation at 12,000 rpm for 5 min. at 4° C.

c. β-galacosidase Assay

The β-galactosidase assay mixture contained 1 mM MgC1₂, 45 mMβ-mercaptoethanol, 0.8 mg/ml o-nitrophenyl-β-D-galactopyranoside and 66mM sodium phosphate, pH 7.5. After incubating the reaction mixture withthe cell extract at 37° C. for increasing time, the reaction wasterminated by the addition of three volumes of 1M Na₂CO₃, and theoptical density was measured at 420 nm. Assay mixture incubated with-outcell extract was used as a control. The linear range of the reaction wasdetermined to be between 0.1-0.8 OD₄₂₀. One unit of β-galactosidaseactivity is defined as the amount of enzyme that will hydrolyse 3 nmolesof o-nitrophenyl-β-D-galactopyranoside in 1 minute at 37° C.

d. Preparation of Cell Extract for hygromycin phosphotransferase Assay

Cells were washed as described above and resuspended into 20 mM Hepesbuffer, pH 7.3, 100 mM potassium acetate, 5 mM Mg acetate and 2 mMdithiothreitol). Cells were disrupted at 0° C. by six 10 sec bursts inan MSE ultrasonic disintegrator using a microtip probe. Cells wereallowed to cool for 1 min after each ultrasonic burst. The extracts wereclarified by centrifuging for 1 min at 2000 rpm in a microcentrifuge.

e. Hygromycin Phosphotransferase Assay

Enzyme activity was measured by means of the phosphocellulose paperbinding assay as described by Haas and Dowding [(1975). Meth. Enzymol.43:611-628]. The cell extract was upplemented with 0.1 M ammoniumchloride and 1 mM adenosine-γ-³²P-triphosphate (specific activity: 300Ci/mmol). The reaction was initiated by the addition of 0.1 mg/mlhygromycin and incubated for increasing time at 37° C. The reaction wasterminated by heating the samples for 5 min at 75° C. in a water bath,and after removing the precipitated proteins by centrifugation for 5 minin a microcentrifuge, an aliquot of the supernatant was spotted on apiece of Whatman P-81 phosphocellulose paper (2 cm²). After 30 sec atroom temperature the papers are placed into 500 ml of hot (75° C.)distilled water for 3 min. While the radioactive ATP remains in solutionunder these conditions, hygromycin phosphate binds strongly andquantitatively to phosphocellulose. The papers are rinsed 3 times in 500ml of distilled water and the bound radioactivity was measured intoluene scintillation cocktail in a Beckman liquid scintillationcounter. Reaction mixture incubated without added hygromycin served as acontrol.

f. Determination of the Copy-Number of the Heterologous Genes

DNA was prepared from the H1D3 and mM2C1 cells using standardpurification protocols involving SDS lysis of the cells followed byProteinase K treatment and phenol/chloroform extractions. The isolatedDNA was digested with an appropriate restriction endonuclease,fractionated on agarose gels, blotted to nylon filters and hybridizedwith a radioactive probe derived either from the β-galactosidase or thehygromycin phosphotransferase genes. The level of hybridization wasquantified in a Molecular Dynamics Phosphorlmage Analyzer. To controlthe total amount of DNA loaded from the different cells lines, thefilters were reprobed with a single copy gene, and the hydridization ofβ-galactosidase and hygromycin phosphotransferase genes was normalizedto the single copy gene hybridization.

g. Determination of Protein Concentration

The total protein content of the cell extracts was measured by theBradford colorimetric assay using bovine serum albumin as standard.

2. Characterization of the β-galactosidase and HygromycinPhsophotransferase Activity Expressed in H1D3 and mM2C1 Cells

In order to establish quantative conditions, the most important kineticparameters of β-galactosidase and hygromycin phosphotransferase activityhave been studied. The β-galactosidase activity measured with acolorimetric assay was linear between the 0.1-0.8 OD₄₂₀ range both forthe nM2C1 and H1D3 cell lines. The β-galactosidase activity was alsoproportional in both cell lines with the amount of protein added to thereaction mixture within 5-100 μg total protein concentration range. Thehygromycin phosphotransferase activity of nM2C1 and H1D3 cell lines wasalso proportional with the reaction time or the total amount of addedcell extract under the conditions described for the β-galactosidase.

a. Comparison of β-galactosidase Activity of mM2C1 and H1D3 Cell Lines

Cell extracts prepared from logarithmically growing mM2Cl and H1D3 celllines were tested for β-galactosidase activity, and the specificactivities were compared in 10 independent experiments. Theβ-galactosidase activity of H1D3 cell extracts was 440±25 U/mg totalprotein. Under identical conditions the β-galactosidase activity of themM2C1 cell extracts was 4.8 times lower: 92±13 U/mg total protein.

β-galactosidase activities of highly subconfluent, subconfluent andnearly confluent cultures of H1D3 and mM2C1 cell lines were alsocompared. In these experiments different numbers of logarithmic H1D3 andmM2C1 cells were seeded in constant volume of culture medium and grownfor 3 days under standard conditions. No significant difference wasfound in the β-galactosidase specific activities of cell cultures grownat different cell densities, and the ratio of H1D3/mM2C1 β-galactosidasespecific activities was also similar for all three cell densities. Inconfluent, stationary cell cultures of H1D3 or mM2C1 cells, however, theexpression of β-galactosidase significantly decreased due likely tocessation of cell division as a result of contact inhibition.

b. Comparison of Hygromycin Phosphotransferase Activity of H1D3 andmM2C1 Cell Lines

The bacterial hygromycin phosphotransferase is present in amembrane-bound form in H1D3 or mM2C1 cell lines. This follows from theobservation that the hygromycin phosphotransferase activity can becompletely removed by high speed centrifugation of these cell extracts,and the enzyme activity can be recovered by resuspending the high speedpellet.

The ratio of the enzyme's specific activity in H1D3 and mM2C1 cell lineswas similar to that of β-galactosidase activity, i.e., H1D3 cells have4.1 times higher specific activity compared with mM2C1 cells.

c. Hygromycin Phosphotransferase Activity in H1D3 and mM2C1 Cells GrownUnder Non-Selective Conditions

The level of expression of the hygromycin phosphotransferase gene wasmeasured on the basis of quantitation of the specific enzyme activitiesin H1D3 and mM2C1 cell lines grown under non-selective conditions for 30generations. The absence of hygromycin in the medium did not influencethe expression of the hygromycin phosphotransferase gene.

3. Quantitation of the Number of β-galactosidase and HygromycinPhosphotransferase Gene Copies in H1D3 and mM2C1 Cell Lines

As described above, the β-galactosidase and hygromycinphosphotransferase genes are located only within the megachromosome, ormicro-megachromosome in H1D3 and mM2C1 cells. Quantitative analysis ofgenomic Southern blots of DNA isolated from H1D3 and mM2Cl cell lineswith the Phosphorlmage Analyzer revealed that the copy number ofβ-galactosidase genes integrated into the megachromosome isapproximately 10 times higher in H1D3 cells than in mM2C1 cells. Thecopy-number of hygromycin phosphotransferase genes is approximately 7times higher in H1D3 cells than in mM2C1 cells.

4. Summary and Conclusions of Results of Quantitation of HeterologousGene Expression in Cells Containing Megachromosomes or DerivativesThereof

Quantitative determination of β-galactosidase activity of highereukaryotic cells (e.g., H1D3 cells) carrying the bacterialβ-galactosidase gene in heterochromatic megachromosomes confirmed theobserved high-level expression of the integrated bacterial gene detectedby cytological staining methods. It has generally been established inreports of studies of the expression of foreign genes in transgenicanimals that, although transgene expression shows correct tissue anddevelopmental specificity, the level of expression is typically low andshows extensive position-dependent variability (i.e., the level oftransgene expression depends on the site of chromosomal integration). Itis has been assumed that the low-level transgene expression may be dueto the absence of special DNA sequences which can insulate the transgenefrom the inhibitory effect of the surrounding chromatin and promote theformation of active chromatin structure required for efficient geneexpression. Several cis-activing DNA sequence elements have beenidentified that abolish this position-dependent variability, and canensure high-level expression of the transgene locus activing region(LAR) sequences in higher eukaryotes and specific chromatin structure(scs) elements in lower eukaryotes (see, et al Eissenberg and Elgin(1991) Trends in Genet. 7:335-340). If these cis-acting DNA sequencesare absent, the level of transgene expression is low and copy-numberindependent.

Although the bacterial β-galactosidase reporter gene contained in theheterochromatic megachromosomes of H1D3 and mM2C1 cells is driven by apotent eukaryotic promoter-enhancer element, no specific cis-acting DNAsequence element was designed and incorporated into the bacterial DNAconstruct which could function as a boundary element. Thus, thehigh-level β-galactosidase expression measured in these cells is ofsignificance, particularly because the β-galactosidase gene in themegachromosome is located in a long, compact heterochromaticenvironment, which is known to be able to block gene expression. Themegachromosome appears to contain DNA sequence element(s) in associationwith the bacterial DNA sequences that function to override theinhibitory effect of heterochromatin on gene expression.

The specificity of the heterologous gene expression in themegachromosome is further supported by the observation that the level ofβ-galactosidase expression is copy-number dependent. In the H1D3 cellline, which carries a full-size megachromosome, the specific activity ofβ-galactosidase is about 5-fold higher than in mM2C1 cells, which carryonly a smaller, truncated version of the megachromosome. A comparison ofthe number of β-galactosidase gene copies in H1D3 and mM2C1 cell linesby quantitative hybridization techniques confirmed that the expressionof β-galactosidase is copy-number dependent. The number of integratedβ-galactosidase gene copies is approximately 10-fold higher in the H1D3cells than in mM2C1 cells. Thus, the cell line containing the greaternumber of copies of the β-galactosidase gene also yields higher levelsof β-galactosidase activity, which supports the copy-number dependencyof expression. The copy number dependency of the β-galactosidase andhygromycin phosphotransferase enzyme levels in cell lines carryingdifferent derivatives of the megachromosome indicates that neither thechromatin organization surrounding the site of integration of thebacterial genes, nor the heterochromatic environment of themegachromosome suppresses the expression of the genes.

The relative amount of β-galactosidase protein expressed in H1D3 cellscan be estimated based on the V_(max) of this enzyme [500 forhomogeneous, crystallized bacterial β-galactosidase (Naider et al.(1972) Biochemistry 11:3202-3210)] and the specific activity of H1D3cell protein. A V_(max) of 500 means that the homogeneousβ-galactosidase protein hydrolyzes 500 μmoles of substrate per minuteper mg of enzyme protein at 37° C. One mg of total H1D3 cell proteinextract can hydrolyze 1.4 μmoles of substrate per minute at 37° C.,which means that 0.28% of the protein present in the H1D3 cell extractis β-galactosidase.

The hygromycin phosphotransferase is present in a membrane-bound form inH1D3 and mM2C1 cells. The tendency of the enzyme to integrate intomembranes in higher eukaryotic cells may be related to its periplasmiclocalization in prokaryotic cells. The bacterial hygromycinphosphotransferase has not been purified to homogeneity; thus, itsV_(max) has not been determined. Therefore, no estimate can be made onthe total amount of hygromycin phosphotransferase protein expressed inthese cell lines. The 4-fold higher specific activity of hygromycinphosphotransferase in H1D3 cells as compared to mM2C1 cells, however,indicates that its expression is also copy number dependent.

The constant and high level expression of the β-galactosidase gene inH1D3 and mM2C1 cells, particularly in the absence of any selectivepressure for the expression of this gene, clearly indicates thestability of the expression of genes carried in the heterochromaticmegachromosomes. This conclusion is further supported by the observationthat the level of hygromycin phosphotransferase expression did notchange when H1D3 and mM2C1 cells were grown under non-selectiveconditions. The consistent high-level, stable, and copy-number dependentexpression of bacterial marker genes clearly indicates that themegachromosome is an ideal vector system for expression of foreigngenes.

EXAMPLE 7

Summary of Some of the Cell Lines with SATACS and Minichromosomes thathave been Constructed

1. EC3/7-Derived Cell Lines

The LMTK⁻-derived cell line, which is a mouse fibroblast cell line, wastransfected with λCM8 and λgtWESneo DNA [see, EXAMPLE 2] to producetransformed cell lines. Among these, was was EC3/7, deposited at theEuropean Collection of Animal cell Culture (ECACC) under Accession No.90051001 [see, U.S. Pat. No. 5,288,625; see, also Hadlaczky et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 88:8106-8110 and U.S. applicationSer. No. 08/375,271]. This cell line contains the dicentric chromosomewith the neo-centromere. Recloning and selection produced cell linessuch as EC3/7C5, which are cell lines with the stable neo-minichromosomeand the formerly dicentric chromosome [see, FIG. 2C].

2. KE1-2/4 Cells

Fusion of EC3/7 with CHO-K20 cells and selection with G418/HAT producedhybrid cell lines, among these was KE1-2/4, which has been depositedwith the ECACC under Accession No. 96040924. KE1-2/4 is a stable cellline that contains the λneo-chromosome [see, FIG. 2D; see, also U.S.Pat. No. 5,288,625], produced by E-type amplifications. KE1-2/4 has beentransfected with vectors containing λ DNA, selectable markers, such asthe puromycin-resistance gene, and genes of interest, such as p53 andthe anti-HIV ribozyme gene. These vectors target the gene of interestinto the λneo-chromosome by virtue of homologous recombination with theheterologous DNA in the chromosome.

3. C5pMCT53 Cells

The EC3/7C5 cell line has been co-transfected with pH132, pCH110 and λDNA [see, EXAMPLE 2] as well as other constructs. Various clones andsubclones have been selected. For example transformation with aconstruct that includes p53 encoding DNA, produced cells designatedC5pMCT53.

4. TF 1004G24 Cells

As discussed above, cotransfection of EC3/7C5 cells with plasmids[pH132, pCH110 available from Pharmacia, see, also Hall et al. (1983) J.Mol. Appl. Gen. 2:101-109] and with λ DNA [λcl 875 Sam 7 (New EnglandBiolabs)] produced transformed cells. Among these is TF1004G24, whichcontains the DNA encoding the anti-HIV ribozyme in theneo-mini-chromosomes. Recloning of TF1004G24 produced numerous celllines. Among these is the NHHL24 cell line. This cell line also has theanti-HIV ribozyme in the neo-minichromosome and expresses high levels ofβ-gal. It has been fused with CHO-K20 cells to produce various hybrids.

5. TF1004G19-Derived Cells

Recloning and selection of the TF1004G transformants produced the cellline TF1004G19, discussed above in EXAMPLE 4, which contains theunstable sausage chromosome and the neo-minichromosome. Single cellcloning produced the TF1004G-19C5 [see FIG. 4] cell line, which has astable sausage chromosome and the neo-minchromosome. TF1004G-19C5 hasbeen fused with CHO cells and the hybrids grown under selectiveconditions to produce the 19C5×Ha4 and 19C5×Ha3 cell lines [see, EXAMPLE4] and others. Recloning of the 19C5×Ha3 cell line yielded a cell linecontaining a gigachromosome, i.e., cell line 19C5×Ha47, see FIG. 2E.BrdU treatment of 19C5×Ha4 cells and growth under selective conditions[neomycin (G) and/or hygromycin (H)] has produced hybrid cell lines suchas the G3D5 and G4D6 cell lines and others. G3D5 has theneo-minichromosome and the megachromosome. G4D6 has only theneo-minichromosome.

Recloning of 19C5×Ha4 cells in H medium produced numerous clones. Amongthese is H1D3 [see FIG. 4], which has the stable megachromosome.Repeated BrdU treatment and reckoning of H1D3 cells has produced theHB31 cell line, which has been used for transformations with thepTEMPUD, pTEMPU, pTEMPU3, and pCEPUR-132 vectors [see, Examples 12 and14, below].

H1D3 has been fused with a CD4⁺ Hela cell line that carries DNA encodingCD4 and neomycin resistance on a plasmid [see, e.g., U.S. Pat. Nos.5,413,914, 5,409,810, 5,266,600, 5,223,263, 5,215,914 and 5,144,019,which describe these Hela cells]. Selection with GH has producedhybrids, including H1×HE41 [see FIG. 4], which carries themegachromosome and also a single human chromosome that includes theCD4neo construct. Repeated BrdU treatment and single cell cloning hasproduced cell lines with the megachromosome [cell line 1B3, see FIG. 4].About 25% of the 1B3 cells have a truncated megachromosome [˜90-120 Mb].Another of these subclones, designated 2C5, was cultured onhygromycin-containing medium and megachromosome-free cell lines wereobtained and grown in G418-containing medium. Recloning of these cellsyielded cell lines such as IB4 and others that have a dwarfmegachromosome [˜150-200 Mb], and cell lines, such as I1C3 and mM2C1,which have a micro-megachromosome [˜50-90 Mb]. The micro-megachromosomeof cell line mM2C1 has no telomeres; however, if desired, synthetictelomeres, such as those described and generated herein, may be added tothe mM2C1 cell micro-megachromosomes. Cell lines containing smallertruncated megachromosomes, such as the mM2C1 cell line containing themicro-megachromosome, can be used to generate even smallermegachromosomes, e.g., ˜10-30 Mb in size. This may be accomplished, forexample, by breakage and fragmentation of the micro-megachromosome inthese cells through exposing the cells to X-ray irradiation, BrdU ortelomere-directed in vivo chromosome fragmentation.

EXAMPLE 8

Replication of the Megachromosome

The homogeneous architecture of the megachromomes provides a uniqueopportunity to perform a detailed analysis of the replication of theconstitutive heterochromatin.

A. Materials and Methods

1. Culture of Cell Lines

H1D3 mouse-hamster hybrid cells carrying the megachromosome [see,EXAMPLE 4] were cultured in F-12 medium containing 10% fetal calf serum[FCS] and 400 μg/ml Hygromycin B [Calbiochem]. G3D5 hybrid cells [see,Example 4] were maintained in F-12 medium containing 10% FCS, 400 μg/mlHygromycin B (Calbiochem), and 400 μg/ml G418 [SIGMA]. Mouse A9fibroblast cells were cultured in F-12 medium supplemented with 10% FCS.

2. BrdU Labelling

In typical experiments, 20-24 parallel semi-confluent cell cultures wereset up in 10 cm Petri dishes. Bromodeoxyuridine (BrdU) (Fluka) wasdissolved in distilled water alkalized with a drop of NaOH, to make a10⁻² M stock solution. Aliquots of 10-50 μl of this BrdU stock solutionwere added to each 10 ml culture, to give a final BrdU concentration of10-50 μM. The cells were cultured in the presence of BrdU for 30 min,and then washed with warm complete medium, and incubated without BrdUuntil required. At this point, 5 μg/ml colchicine was added to a sampleculture every 1 or 2 h. After 1-2 h colchicine treatment, mitotic cellswere collected by “shake-off” and regular chromosome preparations weremade for immunolabelling.

3. Immunolabelling of Chromosomes and in situ Hybridization

Immunolabelling with fluorescein-conjugated anti-BrdU monoclonalantibody (Boehringer) was done according to the manufacturer'srecommendations, except that for mouse A9 chromosomes, 2 M hydrochloricacid was used at 37° C. for 25 min, while for chromosomes of hybridcells, 1 M hydrochloric acid was used at 37° C. for 30 min. In situhybridization with biotin-labelled probes, and indirectimmunofluorescence and in situ hybridization on the same preparation,were performed as described previously [Hadlaczky et al. (1991) Proc.Natl. Acad. Sci. U.S.A. 88:8106-8110, see, also U.S. Pat. No.5,288,6251.

4. Microscopy

All observations and microphotography were made by using a Vanox AHBS(Olympus) microscope. Fujicolor 400 Super G or Fujicolor 1600 Super HGhigh-speed colour negatives were used for photographs.

B. Results

The replication of the megachromosome was analyzed by BrdU pulselabelling followed by immunolabelling. The basic parameters for DNAlabelling in vivo were first established. Using a 30-min pulse of 50 μMBrdU in parallel cultures, samples were taken and fixed at 5 minintervals from the beginning of the pulse, and every 15 min up to 1 hafter the removal of BrdU. Incorporated BrdU was detected byimmunolabelling with fluorescein-conjugated anti-BrdU monoclonalantibody. At the first time point (5 min) 38% of the nuclei werelabelled, and a gradual increase in the number of labelled nuclei wasobserved during incubation in the presence of BrdU, culminating in 46%in the 30-min sample, at the time of the removal of BrdU. At furthertime points (60, 75, and 90 min) no significant changes were observed,and the fraction of labelled nuclei remained constant [44.5-46%].

These results indicate that (i) the incorporation of the BrdU is a rapidprocess, (ii) the 30 min pulse-time is sufficient for reliable labellingof S-phase nuclei, and (iii) the BrdU can be effectively removed fromthe cultures by washing.

The length of the cell cycle of the H1D3 and G3D5 cells was estimated bymeasuring the time between the appearance of the earliest BrdU signalson the extreme late replicating chromosome segments and the appearanceof the same pattern only on one of the chromatids of the chromosomesafter one completed cell cycle. The length of G2 period was determinedby the time of the first detectable BrdU signal on prophase chromosomesand by the labelled mitoses method [Qastler et al. (1959) Exp. Cell Res.17:420-438]. The length of the S-phase was determined in three ways: (i)on the basis of the length of cell cycle and the fraction of nucleilabelled during the 30-120 min pulse; (ii) by measuring the time betweenthe very end of the replication of the extreme late replicatingchromosomes and the detection of the first signal on the chromosomes atthe beginning of S phase; (iii) by the labelled mitoses method. Inrepeated experiments, the duration of the cell cycle was found to be22-26 h, the S phase 10-14 h, and the G2 phase 3.5-4.5 h.

Analyses of the replication of the megachromosome were made in parallelcultures by collecting mitotic cells at two hour intervals following twohours of colchicine treatment. In a repeat experiment, the same analysiswas performed using one hour sample intervals and one hour colchicinetreatment. Although the two procedures gave comparable results, the twohour sample intervals were viewed as more appropriate sinceapproximately 30% of the cells were found to have a considerably shorteror longer cell cycle than the average. The characteristic replicationpatterns of the individual chromosomes, especially some of the latereplicating hamster chromosomes, served as useful internal markers forthe different stages of S-phase. To minimize the error caused by thedifferent lengths of cell cycles in the different experiments, sampleswere taken and analyzed throughout the whole cell cycle until theappearance of the first signals on one chromatid at the beginning of thesecond S-phase.

The sequence of replication in the megachromosome is as follows. At thevery beginning of the S-phase, the replication of the megachromosomestarts at the ends of the chromosomes. The first initiation ofreplication in an interstitial position can usually be detected at thecentromeric region. Soon after, but still in the first quarter of theS-phase, when the terminal region of the short arm has almost completedits replication, discrete initiation signals appear along the chromosomearms. In the second quarter of the S-phase, as replication proceeds, theBrdU-labelled zones gradually widen, and the checkered pattern of themegachromosome becomes clear [see, e.g., FIG. 2F]. At the same time,pericentric regions of mouse chromosomes also show intense incorporationof BrdU. The replication of the megachromosome peaks at the end of thesecond quarter and in the third quarter of the S-phase. At the end ofthe third quarter, and at the very beginning of the last quarter of theS-phase, the megachromosome and the pericentric heterochromatin of themouse chromosomes complete their replication. By the end of S-phase,only the very late replicating segments of mouse and hamster chromosomesare still incorporating BrdU.

The replication of the whole genome occurs in distinct phases. Thesignal of incorporated BrdU increased continuously until the end of thefirst half of the S-phase, but at the beginning of the third quarter ofthe S-phase chromosome segments other than the heterochromatic regionshardly incorporated BrdU. In the last quarter of the S-phase, the BrdUsignals increased again when the extreme late replicating segmentsshowed very intense incorporation.

Similar analyses of the replication in mouse A9 cells were performed ascontrols. To increase the resolution of the immunolabelling pattern,pericentric regions of A9 chromosomes were decondensed by treatment withHoechst 33258. Because of the intense replication of the surroundingeuchromatic sequences, precise localization of the initial BrdU signalin the heterochromatin was normally difficult, even on undercondensedmouse chromosomes. On those chromosomes where the initiation signal(s)were localized unambiguously, the replication of the pericentricheterochromatin of A9 chromosomes was similar to that of themegachromosome. Chromosomes of A9 cells also exhibited replicationpatterns and sequences similar to those of the mouse chromosomes in thehybrid cells. These results indicate that the replicators of themegachromosome and mouse chromosomes retained their original timing andspecificity in the hybrid cells.

By comparing the pattern of the initiation sites obtained after BrdUincorporation with the location of the integration sites of the“foreign” DNA in a detailed analysis of the first quarter of theS-phase, an attempt was made to identify origins of replication(initiation sites) in relation to the amplicon structure of themegachromosome. The double band of integrated DNA on the long arm of themegachromosome served as a cytological marker. The results showed acolocalization of the BrdU and in situ hybridization signals found atthe cytological level, indicating that the “foreign” DNA sequences arein close proximity to the origins of replication, presumably integratedinto the non-satellite sequences between the replicator and thesatellite sequences [see, FIG. 3]. As described in Example 6.B.4, therDNA sequences detected in the megachromosome are also localized at theamplicon borders at the site of integration of the “foreign” DNAsequences, suggesting that the origins of replication responsible forinitiation of replication of the megachromosome involve rDNA sequences.In the pericentric region of several other chromosomes, dot-like BrdUsignals can also be observed that are comparable to the initiationsignals on the megachromosome. These signals may represent similarinitiation sites in the heterochromatic regions of normal chromosomes.

At a frequency of 10⁻⁴, “uncontrolled” amplification of the integratedDNA sequences was observed in the megachromosome. Consistent with theassumption (above) that “foreign” sequences are in proximity of thereplicators, this spatially restricted amplification is likely to be aconsequence of uncontrolled repeated firings of the replicationorigin(s) without completing the replication of the whole segment.

C. Discussion

It has generally been thought that the constitutive heterochromatin ofthe pericentric regions of chromosomes is late replicating [see, e.g.,Miller (1976) Chromosoma 55:165-170]. On the contrary, these experimentsevidence that the replication of the heterochromatic blocks starts at adiscrete initiation site in the first half of the S-phase and continuesthrough approximately three-quarters of S-phase. This difference can beexplained in the following ways: (i) in normal chromosomes, activelyreplicating euchromatic sequences that surround the satellite DNAobscure the initiation signals, and thus the precise localization ofinitiation sites is obscured; (ii) replication of the heterochromatincan only be detected unambiguously in a period during the second half ofthe S-phase, when the bulk of the heterochromatin replicates and mostother chromosomal regions have already completed their replication, orhave not yet started it. Thus, low resolution cytological techniques,such as analysis of incorporation of radioactively labelled precursorsby autoradiography, only detect prominent replication signals in theheterochromatin in the second half of S-phase, when adjacent euchromaticsegments are no longer replicating.

In the megachromosome, the primary initiation sites of replicationcolocalize with the sites where the “foreign” DNA sequences and rDNAsequences are integrated at the amplicon borders. Similar initiationsignals were observed at the same time in the pericentricheterochromatin of some of the mouse chromosomes that do not have“foreign” DNA, indicating that the replication initiation sites at theborders of amplicons may reside in the non-satellite flanking sequencesof the satellite DNA blocks. The presence of a primary initiation siteat each satellite DNA doublet implies that this large chromosome segmentis a single huge unit of replication [megareplicon] delimited by theprimary initiation site and the termination point at each end of theunit. Several lines of evidence indicate that, within this higher-orderreplication unit, “secondary” origins and replicons contribute to thecomplete replication of the megareplicon:

1. The total replication time of the heterochromatic regions of themegachromosome was ˜9-11 h. At the rate of movement of replicationforks, 0.5-5 kb per minute, that is typical of eukaryotic chromosomes[Kornberg et al. (1992) DNA Replication. 2nd. ed., New York: W.H.Freeman and Co, p. 474], replication of a ˜15 Mb replicon would require50-500 h. Alternatively, if only a single replication origin was used,the average replication speed would have to be 25 kb per minute tocomplete replication within 10 h. By comparing the intensity of the BrdUsignals on the euchromatic and the heterochromatic chromosome segments,no evidence for a 5- to 50-fold difference in their replication speedwas found. 2. Using short BrdU pulse labelling, a single origin ofreplication would produce a replication band that moves along thereplicon, reflecting the movement of the replication fork. In contrast,a widening of the replication zone that finally gave rise to thecheckered pattern of the megachromosome was observed, and within thereplication period, the most intensive BrdU incorporation occurred inthe second half of the S-phase. This suggests that once themegareplicator has been activated, it permits the activation and firingof “secondary” origins, and that the replication of the bulk of thesatellite DNA takes place from these secondary origins during the secondhalf of the S-phase. This is supported by the observation that incertain stages of the replication of the megachromosome, the wholeamplicon can apparently be labelled by a short BrdU pulse.

Megareplicators and secondary replication origins seem to be understrict temporal and spatial control. The first initiation within themegachromosomes usually occurred at the centromere, and shortlyafterward all the megareplicators become active. The last segment of themegachromosome to complete replication was usually the second segment ofthe long arm. Results of control experiments with mouse A9 chromosomesindicate that replication of the heterochromatin of mouse chromosomescorresponds to the replication of the megachromosome amplicons.Therefore, the pre-existing temporal control of replication in theheterochromatic blocks is preserved in the megachromosome. Positive[Hassan et al. (1994) J. Cell. Sci. 107:425-4341 and negative [Haase etal. (1994) Mol. Cell. Biol. 14:2516-2524] correlations betweentranscriptional activity and initiation of replication have beenproposed. In the megachromosome, transcription of the integrated genesseems to have no effect on the original timing of the replicationorigins. The concerted, precise timing of the megareplicator initiationsin the different amplicons suggests the presence of specific, cis-actingsequences, origins of replication.

Considering that pericentric heterochromatin of mouse chromosomescontains thousands of short, simple repeats spanning 7-15 Mb, and thecentromere itself may also contain hundreds of kilobases, the existenceof a higher-order unit of replication seems probable. The observeduncontrolled intrachromosomal amplification restricted to a replicationinitiation region of the megachromosome is highly suggestive of arolling-circle type amplification, and provides additional evidence forthe presence of a replication origin in this region.

The finding that a specific replication initiation site occurs at theboundaries of amplicons suggests that replication might play a role inthe amplification process. These results suggest that each amplicon ofthe megachromosome can be regarded as a huge megareplicon defined by aprimary initiation site [megareplicator] containing “secondary” originsof replication. Fusion of replication bubbles from different origins ofbi-directional replication [DePamphilis (1993) Ann. Rev. Biochem.62:29-63] within the megareplicon could form a giant replication bubble,which would correspond to the whole megareplicon. In the light of this,the formation of megabase-size amplicons can be accommodated by areplication-directed amplification mechanism. In H and E-typeamplifications, intrachromosomal multiplication of the amplicons wasobserved [see, above EXAMPLES], which is consistent with the unequalsister chromatid exchange model. Induced or spontaneous unscheduledreplication of a megareplicon in the constitutive heterochromatin mayalso form new amplicon(s) leading to the expansion of the amplificationor to the heterochromatic polymorphism of “normal” chromosomes. The“restoration” of the missing segment on the long arm of themegachromosome may well be the result of the re-replication of oneamplicon limited to one strand.

Taken together, without being bound by any theory, areplication-directed mechanism is a plausible explanation for theinitiation of large-scale amplifications in the centromeric regions ofmouse chromosomes, as well as for the de novo chromosome formations. Ifspecific [amplificator, i.e., sequences controlling amplification]sequences play a role in promoting the amplification process, sequencesat the primary replication initiation site [megareplicator] of themegareplicon are possible candidates.

The presence of rRNA gene sequence at the amplicon borders near theforeign DNA in the megachromosome suggests that this sequencecontributes to the primary replication initiation site and participatesin large-scale amplification of the pericentric heterochromatin in denovo formation of SATACs. Ribosomal RNA genes have an intrinsicamplification mechanism that provides for multiple copies of tandemgenes. Thus, for purposes herein, in the construction of SATACs incells, rDNA will serve as a region for targeted integration, and ascomponents of SATACs constructed in vitro.

EXAMPLE 9

Generation of Chromosomes with Amplified Regions Derived from MouseChromosome 1

To show that the events described in EXAMPLES 2-7 are not unique tomouse chromosome 7 and to show that the EC7/3 cell line is not requiredfor formation of the artificial chromosomes, the experiments have beenrepeated using different initial cell lines and DNA fragments. Any cellor cell line should be amenable to use or can readily be determined thatit is not.

A. Materials

The LP11 cell line was produced by the “scrape-loading” transfectionmethod [Fechheimer et al. (1987) Proc. Natl. Acad. Sci. U.S.A.84:8463-8467] using 25 μg plasmid DNA for 5×10⁶ recipient cells. LP11cells were maintained in F-12 medium containing 3-15 μg/ml Puromycin[SIGMA].

B. Amplification in LP11 Cells

The large-scale amplification described in the above Examples is notrestricted to the transformed EC3/7 cell line or to the chromosome 7 ofmouse. In an independent transformation experiment, LMTK⁻ cells weretransfected using the calcium phosphate precipitation procedure with aselectable puromycin-resistance gene-containing construct designatedpPuroTel [see Example 1.E.2. for a description of this plasmid], toestablish cell line LP11. Cell line LP11 carries chromosome(s) withamplified chromosome segments of different lengths [ ˜150-600 Mb].Cytological analysis of the LP11 cells indicated that the amplificationoccurred in the pericentric region of the long arm of a submetacentricchromosome formed by Robertsonian translocation. This chromosome arm wasidentified by G-banding as chromosome 1. C-banding and in situhybridization with mouse major satellite DNA probe showed that an E-typeamplification had occurred: the newly formed region was composed of anarray of euchromatic chromosome segments containing different amounts ofheterochromatin. The size and C-band pattern of the amplified segmentswere heterogeneous. In several cells, the number of these amplifiedunits exceeded 50; single-cell subclones of LP11 cell lines, however,carry stable marker chromosomes with 10-15 segments and constant C-bandpatterns.

Sublines of the thymidine kinase-deficient LP11 cells (e.g., LP11-15P1C5/7 cell line) established by single-cell cloning of LP11 cells weretransfected with a thymidine kinase gene construct. Stable TK⁺transfectants were established.

EXAMPLE 10

Isolation of SATACS and Other Chromosomes with Atypical Base Contentand/or Size

I. Isolation of Artificial Chromosomes from Endogenous Chromosomes

Artificial chromosomes, such as SATACs, may be sorted from endogenouschromosomes using any suitable procedures, and typically involveisolating metaphase chromosomes, distinguishing the artificialchromosomes from the endogenous chromosomes, and separating theartificial chromosomes from endogenous chromosomes. Such procedures willgenerally include the following basic steps: (1) culture of a sufficientnumber of cells (typically about 2×10⁷ mitotic cells) to yield,preferably on the order of 1×10⁶ artificial chromosomes, (2) arrest ofthe cell cycle of the cells in a stage of mitosis, preferrablymetaphase, using a mitotic arrest agent such as colchicine, (3)treatment of the cells, particularly by swelling of the cells inhypotonic buffer, to increase susceptibility of the cells to disruption,(4) by application of physical force to disrupt the cells in thepresence of isolation buffers for stabilization of the releasedchromosomes, (5) dispersal of chromosomes in the presence of isolationbuffers for stabilization of free chromosomes, (6) separation ofartificial from endogenous chromosomes and (7) storage (and shipping ifdesired) of the isolated artificial chromosomes in appropriate buffers.Modifications and variations of the general procedure for isolation ofartificial chromosomes, for example to accommodate different cell typeswith differing growth characteristics and requirements and to optimizethe duration of mitotic block with arresting agents to obtain thedesired balance of chromosome yield and level of debris, may beempirically determined.

Steps 1-5 relate to isolation of metaphase chromosomes. The separationof artificial from endogenous chromosomes (step 6) may be accomplishedin a variety of ways. For example, the chromosomes may be stained withDNA-specific dyes such as Hoeschst 33258 and chromomycin A₃ and sortedinto artificial and endogenous chromosomes on the basis of dye contentby employing fluorescence-activated cell sorting (FACS). To facilitatelarger scale isolation of the artificial chromosomes, differentseparation techiniques may be employed such as swinging bucketcentrifugation (to effect separation based on chromosome size anddensity) [see, e.g., Mendelsohn et al. (1968) J. Mol. Biol. 32:101-108],zonal rotor centrifugation (to effect separation on the basis ofchromosome size and density) [see, e.g., Burki et al. (1973) Prep.Biochem. 3:157-182; Stubblefield et al. (1978) Biochem. Biophys. Res.Commun. 83:1404-1414, velocity sedimentation (to effect separation onthe basis of chromosome size and shape) [see e.g., Collard et al. (1984)Cytometry 5:9-19]. Immuno-affinity purification may also be employed inlarger scale artificial chromosome isolation procedures. In thisprocess, large populations of artificial chromosome-containing cells(asynchronous or mitotically enriched) are harvested en masse and themitotic chromosomes (which can be released from the cells using standardprocedures such as by incubation of the cells in hypotonic buffer and/ordetergent treatment of the cells in conjunction with physical disruptionof the treated cells) are enriched by binding to antibodies that arebound to solid state matrices (e.g. column resins or magnetic beads).Antibodies suitable for use in this procedure bind to condensedcentromeric proteins or condensed and DNA-bound histone proteins. Forexample, autoantibody LU851 (see Hadlaczky et al. (1989) Chromosoma97:282-288), which recognizes mammalian centromeres may be used forlarge-scale isolation of chromosomes prior to subsequent separation ofartificial from endogenous chromosomes using methods such as FACS. Thebound chromosomes would be washed and eventually eluted for sorting.Immunoaffinity purification may also be used directly to separateartificial chromosomes from endogenous chromosomes. For example, SATACsmay be generated in or transferred to (e.g., by microinjection ormicrocell fusion as described herein) a cell line that has chromosomesthat contain relatively small amounts of heterochromatin, such ashamster cells (e.g., V79 cells or CHO-K1 cells). The SATACs, which arepredominantly heterochromatin, are then separated from the endogenouschromosomes by utilizing anti-heterochromatin binding protein(Drosophila HP-1) antibody conjugated to a solid matrix. Such matrixpreferentially binds SATACs relative to hamster chromosomes. Unboundhamster chromosomes are washed away from the matrix and the SATACs areeluted by standard techniques.

A. Cell Lines and Cell Culturing Procedures

In one isolation procedure, 1B3 mouse-hamster-human hybrid cells [see,FIG. 4] carrying the megachromosome or the truncated megachromosome weregrown in F-12 medium supplemented with 10% fetal calf serum, 150 μg/mlhygromycin B and 400 μg/ml G418. GHB42 [a cell line recloned from G3D5cells] mouse-hamster hybrid cells carrying the megachromosome and theminichromosome were also cultured in F-12 medium containing 10% fetalcalf serum, 150 μg/ml hygromycin B and 400 μg/ml G418. The doubling timeof both cell lines was about 24-40 hours, typically about 32 hours.

Typically, cell monolayers are passaged when they reach about 60-80%confluence and are split every 48-72 hours. Cells that reach greaterthan 80% confluence senesce in culture and are not preferred forchromosome harvesting. Cells may be plated in 100-200 100-mm dishes atabout 50-70% confluency 12-30 hours before mitotic arrest (see, below).

Other cell lines that may be used as hosts for artificial chromosomesand from which the artificial chromosomes may be isolated include, butare not limited to, PtK1 (NBL-3) marsupial kidney cells (ATCC accessionno. CCL35), CHO-K1 Chinese hamster ovary cells (ATCC accession no.CCL61), V79-4 Chinese hamster lung cells (ATCC accession no. CCL93),Indian muntjac skin cells (ATCC accession no. CCL157), LMTK(−) thymidinekinase deficient murine L cells (ATCC accession no. CCL1.3), Sf9 fallarmyworm (Spodoptera frugiperda) ovary cells (ATCC accession no. CRL 1711) and any generated heterokaryon (hybrid) cell lines, such as, forexample, the hamster-murine hybrid cells described herein, that may beused to construct MACs, particularly SATACs.

Cell lines may be selected, for example, to enhance efficiency ofartificial chromosome production and isolation as may be desired inlarge-scale production processes. For instance, one consideration inselecting host cells may be the artificial chromosome-to-totalchromosome ratio of the cells. To facilitate separation of artificialchromosomes from endogenous chromosomes, a higher artificialchromosome-to-total chromosome ratio might be desirable. For example,for H1D3 cells (a murine/hamster heterokaryon; see FIG. 4), this ratiois 1:50, i.e., one artificial chromosome (the megachromosome) to 50total chromosomes. In contrast, Indian muntjac skin cells (ATCCaccession no. CCL157) contain a smaller total number of chromosomes (adiploid number of chromosomes of 7), as do kangaroo rat cells (a diploidnumber of chromosomes of 12) which would provide for a higher artificialchromosome-to-total chromosome ratio upon introduction of, or generationof, artificial chromosomes in the cells.

Another consideration in selecting host cells for production andisolation of artificial chromosomes may be size of the endogenouschromosomes as compared to that of the artificial chromosomes. Sizedifferences of the chromosomes may be exploited to facilitate separationof artificial chromosomes from endogenous chromosomes. For example,because Indian muntjac skin cell chromosomes are considerably largerthan minichromosomes and truncated megachromosomes, separation of theartificial chromosome from the muntjac chromosomes may possibly beaccomplished using univariate (one dye, either Hoechst 33258 orChromomycin A3) FACS separation procedures.

Another consideration in selecting host cells for production andisolation of artificial chromosomes may be the doubling time of thecells. For example, the amount of time required to generate a sufficientnumber of artificial chromosome-containing cells for use in proceduresto isolate artificial chromosomes may be of significance for large-scaleproduction. Thus, host cells with shorter doubling times may bedesirable. For instance, the doubling time of V79 hamster lung cells isabout 9-10 hours in comparison to the approximately 32-hour doublingtime of H1D3 cells.

Accordingly, several considerations may go into the selection of hostcells for the production and isolation of artificial chromosomes. It maybe that the host cell selected as the most desirable for de novoformation of artificial chromosomes is not optimized for large-scaleproduction of the artificial chromosomes generated in the cell line. Insuch cases, it may be possible, once the artificial chromosome has beengenerated in the initial host cell line, to transfer it to a productioncell line more well suited to efficient, high-level production andisolation of the artificial chromosome. Such transfer may beaccomplished through several methods, for example through microcellfusion, as described herein, or microinjection into the production cellline of artificial chromosomes purified from the generating cell lineusing procedures such as described herein. Production cell linespreferably contain two or more copies of the artificial artificialchromosome per cell.

B. Chromosome Isolation

In general, cells are typically cultured for two generations atexponential growth prior to mitotic arrest. To accumulate mitotic 1B3and GHB42 cells in one particular isolation procedure, 5 μg/mlcolchicine was added for 12 hours to the cultures. The mitotic indexobtained was 60-80%. The mitotic cells were harvested by selectivedetachment by gentle pipetting of the medium on the monolayer cells. Itis also possible to utilize mechanical shake-off as a means of releasingthe rounded-up (mitotic) cells from the plate. The cells were sedimentedby centrifugation at 200×g for 10 minutes.

Cells (grown on plastic or in suspension) may be arrested in differentstages of the cell cycle with chemical agents other than colchicine,such as hydroxyurea, vinblastine, colcemid or aphidicolin. Chemicalagents that arrest the cells in stages other than mitosis, such ashydroxyurea and aphidicolin, are used to synchronize the cycles of allcells in the population and then are removed from the cell medium toallow the cells to proceed, more or less simultaneously, to mitosis atwhich time they may be harvested to disperse the chromosomes. Mitoticcells could be enriched for a mechanical shake-off (adherent cells). Thecell cycles of cells within a population of MAC-containing cells mayalso be synchronized by nutrient, growth factor or hormone deprivationwhich leads to an accumulation of cells in the G₁ or G₀ stage;readdition of nutrients or growth factors then allows the quiescentcells to re-enter the the cell cycle in synchrony for abot onegeneration. Cell lines that are known to respond to hormone deprivationin this manner, and which are suitable as hosts for artificialchromosomes, include the Nb2 rat lymphoma cell line which is absolutelydependent on prolactin for stimulation of proliferation (see Gout et al.(1980) Cancer Res. 40:2433-2436). Culturing the cells inprolactin-deficient medium for 18-24 hours leads to arrest ofproliferation, with cells accumulating early in the G₁ phase of the cellcycle. Upon addition of prolactin, all the cells progress through thecell cycle until M phase at which point greater than 90% of the cellswould be in mitosis (addition of colchicine could increase the amount ofthe mitotic cells to greater than 95%). The time between reestablishingproliferation by prolactin addition and harvesting mitotic cells forchromosome separation may be empirically determined.

Alternatively, adherent cells, such as V79 cells, may be grown in rollerbottles and mitotic cells released from the plastic surface by rotatingthe roller bottles at 200 rpm or greater (Shwarchuk et al. (1993) Int.J. Radiat. Biol. 64:601-612). At any given time, approximately 1% of thecells in an exponentially growing asynchronous population is in M-phase.Even without the addition of colchicine, 2×10⁷ mitotic cells have beenharvested from four 1750-cm² roller bottles after a 5-min spin at 200rpm. Addition of colchicine for 2 hours may increase the yield to 6×10⁸mitotic cells.

Several procedures may be used to isolate metaphase chromosomes fromthese cells, including, but not limited to, one based on a polyaminebuffer system [Cram et al. (1990) Methods in Cell Biology 33:377-382],one on a modified hexylene glycol buffer system [Hadlaczky et al. (1982)Chromosoma 86:643-65], one on a magnesium sulfate buffer system [Van denEngh et al. (1988) Cytometry 9:266-270 and Van den Engh et al. (1984)Cytometry 5:108], one on an acetic acid fixation buffer system [Stoehret al. (1982) Histochemistry 74:57-61], and one on a technique utilizinghypotonic KCl and propidium iodide [Cram et al. (1994) XVII meeting ofthe International Society for Analytical Cytology, October 16-21,Tutorial IV Chromosome Analysis and Sorting with Commerical FlowCytometers; Cram et al. (1990) Methods in Cell Biology 33:376].

1. Polyamine Procedure

In the polyamine procedure that was used in isolating artificialchromosomes from either 1B3 or GHB42 cells, about 10⁷ mitotic cells wereincubated in 10 ml hypotonic buffer (75 mM KCl, 0.2 mM spermine, 0.5 mMspermidine) for 10 minutes at room temperature to swell the cells. Thecells are swollen in hypotonic buffer to loosen the metaphasechromosomes but not to the point of cell lysis. The cells were thencentrifuged at 100×g for 8 minutes, typically at room temperature. Thecell pellet was drained carefully and about 10⁷ cells were resuspendedin 1 ml polyamine buffer [15 mM Tris-HCl, 20 mM NaCl, 80 mM KCl, 2 mMEDTA, 0.5 mM EGTA, 14 mM β-mercaptoethanol, 0.1% digitonin, 0.2 mMSpermine, 0.5 mM spermidine] for physical dispersal of the metaphasechromosomes. Chromosomes were then released by gently drawing the cellsuspension up and expelling it through a 22 G needle attached to a 3 mlplastic syringe. The chromosome concentration was about 1-3×10⁸chromosomes/ml.

The polyamine buffer isolation protocol is well suited for obtaininghigh molecular weight chromosomal DNA [Sillar and Young (1981) J.Histochem. Cytochem. 29:74-78; VanDilla et al. (1986) Biotechnology4:537-552; Bartholdi et al. (1988) In “Molecular Genetics of MammalianCells” (M.Goettsman, ed.), Methods in Enzymology 151:252-267. AcademicPress, Orlando]. The chromosome stabilizing buffer uses the polyaminesspermine and spermidine to stabilize chromosome structure [Blumenthal etal. (1979)J. Cell Biol. 81:255-259; Lalande et al. (1985) Cancer Genet.Cytogenet. 23:151-157] and heavy metals chelators to reduce nucleaseactivity.

The polyamine buffer protocol has wide applicability, however, as withother protocols, the following variables must be optimized for each celltype: blocking time, cell concentration, type of hypotonic swellingbuffer, swelling time, volume of hypotonic buffer, and vortexing time.Chromosomes prepared using this protocol are typically highly condensed.

There are several hypotonic buffers that may be used to swell the cells,for example buffers such as the following: 75 mM KCl; 75 mM KCl, 0.2 mMspermine, 0.5 mM spermidine; Ohnuki's buffer of 16.2 mM sodium nitrate,6.5 mM sodium acetate, 32.4 mM KCl [Ohnuki (1965) Nature 208:916-917 andOhnuki (1968) Chromosoma 25:402-428]; and a variation of Ohnuki's bufferthat additionally contains 0.2 mM spermine and 0.5 mM spermidine. Theamount and hypotonicity of added buffer vary depending on cell type andcell concentration. Amounts may range from 2.5-5.5 ml per 10⁷ cells ormore. Swelling times may vary from 10-90 minutes depending on cell typeand which swelling buffer is used.

The composition of the polyamine isolation buffer may also be varied.For example, one modified buffer contains 15 mM Tris-HCl, pH 7.2, 70 mMNaCl, 80 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 14 mM beta-mercaptoethanol,0.25% Triton-X, 0.2 mM spermine and 0.5 mM spermidine.

Chromosomal dispersal may also be accomplished by a variety of physicalmeans. For example, cell suspension may be gently drawn up and expelledin a 3-ml syringe fitted with a 22-gauge needle [Cram et al. (1990)Methods in Cell Biology 33:377-382], cell suspension may be agitated ona bench-top vortex [Cram et al. (1990) Methods in Cell Biology33:377-382], cell suspension may be disrupted with a homogenizer [Sillarand Young (1981) J. Histochem. Cytochem. 29:74-78; Carrano et al. (1979)Proc. Natl. Acad. Sci. U.S.A. 76:1382-1384] and cell suspension may bedisrupted with a bench-top ultrasonic bath [Stoehr et al. (1982)Histochemistry 74:57-61].

2. Hexylene Glycol Buffer System

In the hexylene glycol buffer procedure that was used in isolatingartificial chromosomes from either 1B3 or GHB42 cells, about 8×10⁶mitotic cells were resuspended in 10 ml glycine-hexylene glycol buffer[100 mM glycine, 1% hexylene glycol, pH 8.4-8.6 adjusted with saturatedCa-hydroxide solution] and incubated for 10 minutes at 37° C., followedby centrifugation for 10 minutes to pellet the nuclei. The supernatantwas centrifuged again at 200×g for 20 minutes to pellet the chromosomes.Chromosomes were resuspended in isolation buffer (1-3×10⁸chromosomes/ml).

The hexylene glycol buffer composition may also be modified. Forexample, one modified buffer contains 25 mM Tris-HCl, pH 7.2, 750 mMhexylene glycol, 0.5 mM CaCl₂, 1.0 mM MgCl₂ [Carrano et al. (1979) Proc.Natl. Acad. Sci. U.S.A. 76:1382-1384].

3. Magnesium-Sulfate Buffer System

This buffer system may be used with any of the methods of cell swellingand chromosomal dispersal, such as described above in connection withthe polyamine and hexylene glycol buffer systems. In this procedure,mitotic cells are resuspended in the following buffer: 4.8 mM HEPES, pH8.0, 9.8 mM MgSO₄, 48 mM KCl, 2.9 mM dithiothreitol [Van den Engh et al.(1985) Cytometry 6:92 and Van den Engh et al. (1984) Cytometry 5:108].

4. Acetic Acid Fixation Buffer System

This buffer system may be used with any of the methods of cell swellingand chromosomal dispersal, such as described above in connection withthe polyamine and hexylene glycol buffer systems. In this procedure,mitotic cells are resuspended in the following buffer: 25 mM Tris-HCl,pH 3.2, 750 mM (1,6)-hexandiol, 0.5 mM CaCl₂, 1.0% acetic acid [Stoehret al. (1982) Histochemistry 74:57-61].

5. KCl-Propidium Iodide Buffer System

This buffer system may be used with any of the methods of cell swellingand chromosomal dispersal, such as described above in connection withthe polyamine and hexylene glycol buffer systems. In this procedure,mitotic cells are resuspended in the following buffer: 25 mM KCl, 50μg/ml propidium iodide, 0.33% Triton X-100, 333 μg/ml RNase [Cram et al.(1990) Methods in Cell Biology 33:376].

The fluorescent dye propidium iodide is used and also serves as achromosome stabilizing agent. Swelling of the cells in the hypotonicmedium (which may also contain propidium iodide) may be monitored byplacing a small drop of the suspension on a microscope slide andobserving the cells by phase/fluorescent microscopy. The cells shouldexclude the propidium iodide while swelling, but some may lyseprematurely and show chromosome fluorescence. After the cells have beencentrifuged and resuspended in the KCl-propidium iodide buffer system,they will be lysed due to the presence of the detergent in the buffer.The chromosomes may then be dispersed and then incubated at 37° C. forup to 30 minutes to permit the RNase to act. The chromosome preparationis then analyzed by flow cytometry. The propidium iodide fluorescencecan be excited at the 488 nm wavelength of an argon laser and detectedthrough an OG 570 optical filter by a single photomultiplier tube. Thesingle pulse may be integrated and acquired in an univariate histogram.The flow cytometer may be aligned to a CV of 2% or less using small (1.5μm diameter) microspheres. The chromosome preparation is filteredthrough 60 μm nylon mesh before analysis.

C. Staining of Chromosomes with DNA-Specific Dyes

Subsequent to isolation, the chromosome preparation was stained withHoechst 33258 at 6 μg/ml and chromomycin A3 at 200 μg/ml. Fifteenminutes prior to analysis, 25 mM Na-sulphite and 10 mM Na-citrate wereadded to the chromosome suspension.

D. Flow Sorting of Chromosomes

Chromosomes obtained from 1B3 and GHB42 cells and maintained weresuspended in a polyamine-based sheath buffer (0.5 mM EGTA, 2.0 mM EDTA,80 mM KCl, 70 mM NaCl, 15 mM Tris-HCl, pH 7.2, 0.2 mM spermine and 0.5mM spermidine) [Sillar and Young (1981) J. Histochem. Cytochem.29:74-78]. The chromosomes were then passed through a dual-laser cellsorter [FACStar Plus or FAXStar Vantage Becton Dickinson ImmunocytometrySystem; other dual-laser sorters may also be used, such as thosemanufactured by Coulter Electronics (Elite ESP) and Cytomation (MoFlo)]in which two lasers were set to excite the dyes separately, allowing abivariate analysis of the chromosome by size and base-pair composition.Because of the difference between the base composition of the SATACs andthe other chromosomes and the resulting difference in interaction withthe dyes, as well as size differences, the SATACs were separated fromthe other chromosomes.

E. Storage of the Sorted Artificial Chromosomes

Sorted chromosomes may be pelleted by centrifugation and resuspended ina variety of buffers, and stored at 4° C. For example, the isolatedartificial chromosomes may be stored in GH buffer (100 mM glycine, 1%hexylene glycol pH 8.4-8.6 adjusted with saturated Ca-hydroxidesolution) [see, e.g., Hadlaczky et al. (1982) Chromosoma 86:643-659] forone day and embedded by centrifugation into agarose. The sortedchromosomes were centrifuged into an agarose bed and the plugs arestored in 500 mM EDTA at 4° C. Additional storage buffers includeCMB-I/polyamine buffer (17.5 mM Tris-HCl, pH 7.4, 1.1 mM EDTA, 50 mMepsilon-amino caproic acid, 5 mM benzamide-HCl, 0.40 mM spermine, 1.0 mMspermidine, 0.25 mM EGTA, 40 mM KCl, 35 mM NaCl) and CMB-II/polyaminebuffer (100 mM glycine, pH 7.5, 78 mM hexylene glycol, 0.1 mM EDTA, 50mM epsilon-amino caproic acid, 5 mM benzamide-HCl, 0.40 mM spermine, 1.0mM spermidine, 0.25 mM EGTA, 40 mM KCl, 35 mM NaCl).

When microinjection is the intended use, the sorted chromosomes arestored in 30% glycerol at −20° C. Sorted chromosomes may also be storedwithout glycerol for short periods of time (3-6 days) in storage buffersat 4° C. Exemplary buffers for microinjection include CBM-I (10 mMTris-HCl, pH 7.5, 0.1 mM EDTA, 50 mM epsilon-amino caproic acid, 5 mMbenzamide-HCl, 0.30 mM spermine, 0.75 mM spermidine), CBM-II (100 mMglycine, pH 7.5, 78 mM hexylene glycol, 0.1 mM EDTA, 50 mM epsilon-aminocaproic acid, 5 mM benzamide-HCl, 0.30 mM spermine, 0.75 mM spermidine).

For long-term storage of sorted chromosomes, the above buffers arepreferably supplemented with 50% glycerol and stored at −20° C.

F. Quality Control

1. Analysis of the Purity

The purity of the sorted chromosomes was checked by fluorescence in situhybridization (FISH) with a biotin-labeled mouse satellite DNA probe[see, Hadlaczky et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:8106-8110]. Purity of the isolated chromosomes was about 97-99%.

2. Characteristics of the Sorted Chromosomes

Pulsed field gel electrophoresis and Southern hybridization were carriedout to determine the size distribution of the DNA content of the sortedartificial chromosomes.

G. Functioning of the Purified Artificial Chromosomes

To check whether their activity is preserved, the purified artificialchromosomes may be microinjected (using methods such as those describedin Example 13) into primary cells, somatic cells and stem cells whichare then analyzed for expression of the heterologous genes carried bythe artificial chromosomes, e.g., such as analysis for growth onselective medium and assays of β-galactosidase activity.

II. Sorting of Mammalian Artificial Chromosome-Containing Microcells

A. Micronucleation

Cells were grown to 80-90% confluency in 4 Tb150 flasks. Colcemid wasadded to a final concentration of 0.06 μg/ml, and then incubated withthe cells at 37° C. for 24 hours.

B. Enucleation

Ten μg/ml cytochalasin B was added and the resulting microcells werecentrifuged at 15,000 rpm for 70 minutes at 28-33° C.

C. Purification of Microcells by Filtration

The microcells were purified using Swinnex filter units and Nucleoporefilters [5 μm and 3 μm].

D. Staining and Sorting Microcells

As above, the cells were stained with Hoechst and chromomycin A3 dyes.The microcells were sorted by cell sorter to isolate the microcells thatcontain the mammalian artificial chromosomes.

E. Fusion

The microcells that contain the artificial chromosome are fused, forexample, as described in Example 1.A.5., to selected primary cells,somatic cells, embryonic stem cells to generate transgenic (non-human)animals and for gene therapy purposes, and to other cells to deliver thechromosomes to the cells.

EXAMPLE 11

Introduction of mammalian artificial chromosomes into insect cellsInsect cells are useful hosts for MACs, particularly for use in theproduction of gene products, for a number of reasons, including:

1. A mammalian artificial chromosome provides an extra-genomic specificintegration site for introduction of genes encoding proteins of interest[reduced chance of mutation in production system].

2. The large size of an artificial chromosome permits megabase size DNAintegration so that genes encoding an entire pathway leading to aprotein or nonprotein of therapeutic value, such as an alkaloid[digitalis, morphine, taxol] can be accomodated by the artificialchromosome.

3. Amplification of genes encoding useful proteins can be accomplishedin the artificial mammalian chromosome to obtain higher protein yieldsin insect cells.

4. Insect cells support required post-translational modifications(glycosylation, phosphorylation) essential for protein biologicalfunction.

5. Insect cells do not support mammalian viruses—eliminatescross-contamination of product with human infectious agents.

6. The ability to introduce chromosomes circumvents traditionalrecombinant baculovirus systems for production of nutritional,industrial or medicinal proteins in insect cell systems.

7. The low temperature optimum for insect cell growth (28° C.) permitsreduced energy cost of production.

8. Serum free growth medium for insect cells will result in lowerproduction costs.

9. Artificial chromosome-containing cells can be stored indefinitely atlow temperature.

10. Insect larvae will serve as biological factories for the productionof nutritional, medicinal or industrial proteins by microinjection offertilized insect eggs.

A. Demonstration that Insect Cells Recognize Mammalian Promoters

Gene constructs containing a mammalian promoter, such as the CMVpromoter, linked to a detectable marker gene [Renilla luciferase gene(see, e.g., U.S. Pat. No. 5,292,658 for a description of DNA encodingthe Renilla luciferase, and plasmid pTZrLuc-1, which can provide thestarting material for construction of such vectors, see also SEQ ID No.10] and also including the simian virus 40 (SV40) promoter operablylinked to the β-galactosidase gene were introduced into the cells of twospecies Trichoplusia ni [cabbage looper] and Bombyx mori [silk worm].

After transferring the constructs into the insect cell lines either byelectroporation or by microinjection, expression of the marker genes wasdetected in luciferase assays (see e.g., Example 12.C.3) and inβ-galactosidase assays (such as lacZ staining assays) after a 24-hincubation. In each case a positive result was obtained in the samplescontaining the genes which was absent in samples in which the genes wereomitted. In addition, a B. mori β-actin promoter-Renilla luciferase genefusion was introduced into the T. ni and B. mori cells which yieldedlight emission after transfection. Thus, certain mammalian promotersfunction to direct expression of these marker genes in insect cells.Therefore, MACs are candidates for expression of heterologous genes ininsect cells.

B. Construction of Vectors for use in Insect Cells and Fusion WithMammalian Cells

1. Transform LMTK⁻ cells with expression vector with:

-   -   a. B. mori β-actin promoter—Hyg^(r) selectable marker gene for        insect cells, and    -   b. SV40 or CMV promoters controlling a puromycin^(r) selectable        marker gene for mammalian cells.    -   2. Detect expression of the mammalian promoter in LMTK cells        (puromycin^(r) LMTK cells)    -   3. Use puromycin^(r) cells in fusion experiments with Bombyx and        Trichoplusia cells, select Hyg^(r) cells.        C. Insertion of the MACs into Insect Cells

These experiments are designed to detect expression of a detectablemarker gene [such as the β-galactosidase gene expressed under thecontrol of a mammalian promoter, such as pSV40] located on a MAC thathas been introduced into an insect cell. Data indicate that β-gal wasexpressed.

Insect cells are fused with mammalian cells containing mammalianartificial chromosomes, e.g., the minichromosome [EC3/7C5] or the miniand the megachromosome [such as GHB42, which is a cell line reclonedfrom G3D5] or a cell line that carries only the megachromosome [such asH1D3 or a redone therefrom]. Fusion is carried out as follows:

-   -   1. mammalian+insect cells (50/50%) in log phase growth are        mixed;    -   2. calcium/PEG cell fusion: (10 min—0.5 h);    -   3. heterokaryons (+72 h) are selected.

The following selection conditions to select for insect cells thatcontain a MAC can be used: [+=positive selection; −=negative selection]:

-   -   1. growth at 28° C. (+insect cells, −mammalian cells);    -   2. Graces insect cell medium [SIGMA] (−mammalian cells);    -   3. no exogenous CO₂ (−mammalian cells); and/or    -   4. antibiotic selection (Hyg or G418) (+transformed insect        cells).

Immediately following the fusion protocol, many heterokaryons [fusionevents] are observed between the mammalian and each species of insectcells [up to 90% heterokaryons]. After growth [2+ weeks] on insectmedium containing G418 and/or hygromycin at selection levels used forselection of transformed mammalian cells, individual colonies aredetected growing on the fusion plates. By virtue of selection for theantibiotic resistance conferred by the MAC and selection for insectcells, these colonies should contain MACs.

The B. mori β-actin gene promoter has been shown to direct expression ofthe β-galactosidase gene in B. mori cells and mammalian cells (e.g.,EC3/7C5 cells). The B. mori β-actin gene promoter is, thus, particularlyuseful for inclusion in MACs generated in mammalian cells that willsubsequently be transferred into insect cells because the presence ofany marker gene linked to the promoter can be determined in themammalian and resulting insect cell lines.

EXAMPLE 12

Preparation of Chromosome Fragmentation Vectors and Other Vectors forTargeted Integration of DNA into MACs

Fragmentation of the megachromosome should ultimately result in smallerstable chromosomes that contain about 15 Mb to 50 Mb that will be easilymanipulated for use as vectors. Vectors to effect such fragmentationshould also aid in determination and identification of the elementsrequired for preparation of an in vitro-produced artificial chromosome.

Reduction in the size of the megachromosome can be achieved in a numberof different ways including: stress treatment, such as by starvation, orcold or heat treatment; treatment with agents that destabilize thegenome or nick DNA, such as BrdU, coumarin, EMS and others; treatmentwith ionizing radiation [see, e.g., Brown (1992) Curr. Opin. Genes Dev.2:479-486]; and telomere-directed in vivo chromosome fragmentation [see,e.g., Farr et al. (1995) EMBO J. 14:5444-5454].

A. Preparation of Vectors for Fragmentation of the Artificial Chromosomeand Also for Targeted Integration of Selected Gene Products

1. Construction of pTEMPUD

Plasmid pTEMPUD [see FIG. 5] is a mouse homologous recombination“killer” vector for in vivo chromosome fragmentation, and also forinducing large-scale amplification via site-specific integration. Withreference to FIG. 5, the ˜3,625-bp SalI-PstI fragment was derived fromthe pBabe-puro retroviral vector [see, Morgenstern et al. (1990) NucleicAcids Res. 18:3587-3596]. This fragment contains DNA encoding ampicillinresistance, the pUC origin of replication, and the puromycin N-acetyltransferase gene under control of the SV40 early promoter. The URA3 geneportion comes from the pYAC5 cloning vector [SIGMA]. URA3 was cut out ofpYAC5 with SalI-XhoI digestion, cloned into pNEB193 [New EnglandBiolabs], which was then cut with EcoRI-SalI and ligated to the SalIsite of pBabepuro to produce pPU.

A 1293-bp fragment [see SEQ ID No. 1] encoding the mouse majorsatellite, was isolated as an EcoRI fragment from a DNA library producedfrom mouse LMTK⁻ fibroblast cells and inserted into the EcoRI site ofpPU to produce pMPU.

The TK promoter-driven diphtheria toxin gene [DT-A] was derived frompMC1DT-A [see, Maxwell et al. (1986) Cancer Res. 46:4660-4666] byBglII-XhoI digestion and cloned into the pMC1neo poly A expressionvector [STRATAGENE, La Jolla, Calif.] by replacing theneomycin-resistance gene coding sequence. The TK promoter, DT-A gene andpoly A sequence were removed from this vector, cohesive ends were filledwith Klenow and the resulting fragment blunt end-ligated and ligatedinto the SnaBI [TACGTA] of pMPU to produce pMPUD.

The Hutel 2.5-kb fragment [see SEQ ID No.3] was inserted at the PstIsite [see the 6100 PstI-3625 PstI fragment on pTEMPUD] of pMPUD toproduce pTEMPUD. This fragment includes a human telomere. It includes aunique BqlII site [see nucleotides 1042-1047 of SEQ ID No.3], which willbe used as a site for introduction of a synthetic telomere that includesmultiple repeats [80] of TTAGGG with BamHI and BglII ends for insertioninto the BglII site which will then remain unique, since the BamHIoverhang is compatible with the BqlII site. Ligation of a BamHI fragmentto a BglII destroys the BglII site, so that only a single BglII sitewill remain. Selection for the unique BilII site insures that thesynthetic telomere will be inserted in the correct orientation. Theunique BglII site is the site at which the vector is linearized.

To generate a synthetic telomere made up of multiple repeats of thesequence TTAGGG, attempts were made to clone or amplify ligationproducts of 30-mer oligonucleotides containing repeats of the sequence.Two 30-mer oligonucleotides, one containing four repeats of TTAGGGbounded on each end of the complete run of repeats by half of a repeatand the other containing five repeats of the complement AATCCC, wereannealed. The resulting double-standed molecule with 3-bp protrudingends, each representing half of a repeat, was expected to ligate withitself to yield concatamers of n×30 bp. However, this approach wasunsuccessful, likely due to formation of quadruplex DNA from the G-richstrand. Similar difficulty has been encountered in attempts to generatelong repeats of the pentameric human satellite II and III units. Thus,it appears that, in general, any oligomer sequence containingperiodically spaced consecutive series of guanine nucleotides is likelyto form undesired quadruplex formation that hinders construction of longdouble-stranded DNAs containing the sequence.

Therefore, in another attempt to construct a synthetic telomere forinsertion into the BglII site of pTEMPUD, the starting material wasbased on the complementary C-rich repeat sequence (i.e., AATCCC) whichwould not be susceptible to quadruplex structure formation. Twoplasmids, designated pTEL280110 and pTel280111, were constructed asfollows to serve as the starting materials.

First, a long oligonucleotide containing 9 repeats of the sequenceAATCCC (i.e., the complement of telomere sequence TTAGGG) in reverseorder bounded on each end of the complete run of repeats by half of arepeat (therefore, in essence, containing 10 repeats), and recognitionsites for PstI and PacI restriction enzymes was synthesized usingstandard methods. The oligonucleotide sequence is as follows: (SEQ IDNO. 29) 5′-AAACTGCAGGTTAATTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCGGGAT-3′

A partially complementary short oligonucleotide of sequence3′-TTGGGCCCTAGGCTTAAGG-5′ (SEQ ID NO. 30)was also synthesized. The oligonucleotides were gel-purified, annealed,repaired with Klenow polymerase and digested with EcoRI and PstI. Theresulting EcoRI/PstI fragment was ligated with EcoRI/PstI-digestedpUC19. The resulting plasmid was used to transform E. coli DH5αcompetent cells and plasmid DNA (pTel102) from one of the transformantssurviving selection on LB/ampicillin was digested with PacI, renderedblunt-ended by Klenow and dNTPs and digested with HindIII. The resulting2.7-kb fragment was gel-purified.

Simultaneously, the same plasmid was amplified by the polymerase chainreaction using extended and more distal 26-mer M13 sequencing primers.The amplification product was digested with SmaI and HindIII, thedouble-stranded 84-bp fragment containing the 60-bp telomeric repeat(plus 24 bp of linker sequence) was isolated on a 6% nativepolyacrylamide gel, and ligated with the double-digested pTel102 toyield a 120-bp telomeric sequence. This plasmid was used to transformDH5α cells. Plasmid DNA from two of the resulting recombinants thatsurvived selection on ampicillin (100 μg/ml) was sequenced on an ABI DNAsequencer using the dye-termination method. One of the plasmids,designated pTel29, contained a sequence of 20 repeats of the sequenceTTAGGG (i.e., 19 successive repeats of TTAGGG bounded on each end of thecomplete run of repeats with half of a repeat). The other plasmid,designated pTel28, had undergone a deletion of 2 bp (TA) at the junctionwhere the two sequences, each containing, in essence, 10 repeats of theTTAGGG sequence, that had been ligated to yield the plasmid. Thisresulted in a GGGTGGG motif at the junction in pTel28. This mutationprovides a useful tag in telomere-directed chromosome fragmentationexperiments. Therefore, the pTel29 insert was amplified by PCR usingpUC/M13 sequencing primers based on sequence somewhat longer and fartherfrom the polylinker than usual as follows:5′-GCCAGGGTTTTCCCAGTCACGACGT-3′ (SEQ ID NO. 31)

or in some experiments 5′-GCTGCAAGGCGATTAAGTTGGGTAAC-3′ (SEQ ID NO. 32)

as the m13 forward primer, and 5′-TATGTTGTGTGGAATTGTGAGCGGAT-3′ (SEQ IDNO. 33)as the m13 reverse primer.The amplification product was digested with SmaI and HindIII. Theresulting 144-bp fragment was gel-purified on a 6% native polyacrylamidegel and ligated with pTel28 that had been digested with PacI,blunt-ended with Klenow and dNTP and then digested with HindIII toremove linker. The ligation yielded a plasmid designated pTel2801containing a telomeric sequence of 40 repeats of the sequence TTAGGG inwhich one of the repeats (i.e., the 30th repeat) lacked two nucleotides(TA), due to the deletion that had occurred in pTel28, to yield a repeatas follows: TGGG.

In the next extension step, pTel2801 was digested with SmaI and HindIIIand the 264-bp insert fragment was gel-purified and ligated withpTel2801 which had been digested with PacI, blunt-ended and digestedwith HindIII. The resulting plasmid was transformed into DH5α cells andplasmid DNA from 12 of the resulting transformants that survivedselection on ampicillin was examined by restriction enzyme analysis forthe presence of a 0.5-kb EcoRI/PstI insert fragment. Eleven of therecombinants contained the expected 0.5-kb insert. The inserts of two ofthe recombinants were sequenced and found to be as expected. Theseplasmids were designated pTel280110 and pTel280111. These plasmids,which are identical, both contain 80 repeats of the sequence TTAGGG, inwhich two of the repeats (i.e., the 30th and 70th repeats) lacked twonucleotides (TA), due to the deletion that had occurred in pTel28, toyield a repeat as follows: TGGG. Thus, in each of the cloning steps(except the first), the length of the synthetic telomere doubled; thatis, it was increasing in size exponentially. Its length was 60×2^(n) bp,wherein n is the number of extension cloning steps undertaken.Therefore, in principle (assuming E. coli, or any other microbial host,e.g., yeast, tolerates long tandem repetitive DNA), it is possible toassemble any desirable size of safe telomeric repeats.

In a further extension step, pTel280110 was digested with PacI,blunt-ended with Klenow polymerase in the presence of dNTP, thendigested with HindIII. The resulting 0.5-kb fragment was gel purified.Plasmid pTel280111 was cleaved with SmaI and HindIII and the 3.2-kbfragment was gel-purified and ligated to the 0.5-kb fragment frompTel280110. The resulting plasmid was used to transform DH5α cells.Plasmid DNA was purified from transformants surviving ampicillinselection. Nine of the selected recombinants were examined byrestriction enzyme analysis for the presence of a 1.0-kb EcoRI/PstIfragment. Four of the recombinants (designated pTlk2, pTlk6, pTlk7 andpTlk8) were thus found to contain the desired 960 bp telomere DNA insertsequence that included 160 repeats of the sequence TTAGGG in which fourof the repeats lacked two nucleotides (TA), due to the deletion that hadoccurred in pTel28, to yield a repeat as follows: TGGG. Partial DNAsequence analysis of the EcoRI/PstI fragment of two of these plasmids(i.e., pTlk2 and pTlk6), in which approximately 300 bp from both ends ofthe fragment were elucidated, confirmed that the sequence was composedof successive repeats of the TTAGGG sequence.

In order to add PmeI and BglII sites to the synthetic telomere sequence,pTlk2 was digested with PacI and PstI and the 3.7-kb fragment (i.e.,2.7-kb pUC19 and 1.0-kb repeat sequence) was gel-purified and ligated atthe PstI cohesive end with the following oligonucleotide5′-GGGTTTAAACAGATCTCTGCA-3′ (SEQ ID NO. 34). The ligation product wassubsequently repaired with Klenow polymerase and dNTP, ligated to itselfand transformed into E. coli strain DH5α. A total of 14 recombinantssurviving selection on ampicillin were obtained. Plasmid DNA from eachrecombinant was able to be cleaved with BqlII indicating that this addedunique restriction site had been retained by each recombinant. Four ofthe 14 recombinants contained the complete 1-kb synthetic telomereinsert, whereas the insert of the remaining 10 recombinants hadundergone deletions of various lengths. The four plasmids in which the1-kb synthetic telomere sequence remained intact were designated pTlkV2,pTlkV5, pTlkV8 an pTlkV12. Each of these plasmids could also be digestedwith PmeI; in addition the presence of both the BglII nad PmeI sites wasverified by sequence analysis. Any of these four plasmids can bedigested with BamHI and BglII to release a fragment containing the 1-kbsynthetic telomere sequence which is then ligated with BqlII-digestedpTEMPUD.

2. Use of pTEMPUD for in vivo Chromosome Fragmentation

Linearization of pTEMPUD by BqlII results in a linear molecule with ahuman telomere at one end. Integration of this linear fragment into thechromosome, such as the megachromosome in hybrid cells or any mousechromosome which contains repeats of the mouse major satellite sequenceresults in integration of the selectable marker puromycin-resistancegene and cleavage of the plasmid by virtue of the telomeric end. The DTgene prevents that entire linear fragment from integrating by randomevents, since upon integration and expression it is toxic. Thus randomintegration will be toxic, so site-directed integration into thetargeted DNA will be selected. Such integration will produce fragmentedchromosomes.

The fragmented truncated chromosome with the new telomere will survive,and the other fragment without the centromere will be lost. Repeated invivo fragmentations will ultimately result in selection of the smallestfunctioning artificial chromosome possible. Thus, this vector can beused to produce minichromosomes from mouse chromosomes, or to fragmentthe megachromosome. In principle, this vector can be used to target anyselected DNA sequence in any chromosome to achieve fragmentation.

3. Construction of pTERPUD

A fragmentation/targeting vector analogous to pTEMPUD for in vivochromosome fragmentation, and also for inducing large-scaleamplification via site-specific integration but which is based on mouserDNA sequence instead of mouse major satellite DNA has been designatedpTERPUD. In this vector, the mouse major satellite DNA sequence ofpTEMPUD has been replaced with a 4770-bp BamHI fragment ofmegachromosome clone 161 which contains sequence corresponding tonucleotides 10,232-15,000 in SEQ ID NO. 16.

4. pHASPUD and pTEMPhu3

Vectors that specifically target human chromosomes can be constructedfrom pTEMPUD. These vectors can be used to fragment specific humanchromosomes, depending upon the selected satellite sequence, to producehuman minichromosomes, and also to isolate human centromeres.

a. pHASPUD

To render pTEMPUD suitable for fragmenting human chromosomes, the mousemajor satellite sequence is replaced with human satellite sequences.Unlike mouse chromosomes, each human chromosome has a unique satellitesequence. For example, the mouse major satellite has been replaced witha human hexameric α-satellite [or alphoid satellite] DNA sequence. Thissequence is an 813-bp fragment [nucleotide 232-1044 of SEQ ID No. 2]from clone pS12, deposited in the EMBL database under Accession numberX60716, isolated from a human colon carcinoma cell line Colo320[deposited under Accession No. ATCC CCL 220.1]. The 813-bp alphoidfragment can be obtained from the pS12 clone by nucleic acidamplification using synthetic primers, each of which contains an EcoRIsite, as follows: forward primer [SEQ ID No. 4] GGGGAATTCAT TGGGATGTTTCAGTTGA reverse primer [SEQ ID No. 5] CGAAAGTCCCC CCTAGGAGAT CTTAAGGA.

Digestion of the amplified product with EcoRI results in a fragment withEcoRI ends that includes the human α-satellite sequence. This sequenceis inserted into pTEMPUD in place of the EcoRI fragment that containsthe mouse major satellite to yield pHASPUD.

Vector pHASPUD was linearized with BqlII and used to transform EJ30(human fibroblast) cells by scrape loading. Twenty-sevenpuromycin-resistant transformant strains were obtained.

b. pTEMPhu3

In pTEMPhu3, the mouse major satellite sequence is replaced by the 3 kbhuman chromosome 3-specific α-satellite from D3Z1 [deposited under ATCCAccession No. 85434; see, also Yrokov (1989) Cytogenet. Cell Genet.51:1114].

5. Use of the pTEMPHU3 to Induce Amplification on Human Chromosome #3

Each human chromosome contains unique chromosome-specific alphoidsequence. Thus, pTEMPHU3, which is targeted to the chromosome 3-specificα-satellite, can be introduced into human cells under selectiveconditions, whereby large-scale amplification of the chromosome 3centromeric region and production of a de novo chromosome ensues. Suchinduced large-scale amplification provides a means for inducing de novochromosome formation and also for in vivo cloning of defined humanchromosome fragments up to megabase size.

For example, the break-point in human chromosome 3 is on the short armnear the centromere. This region is involved in renal cell carcinomaformation. By targeting pTEMPhu3 to this region, the induced large-scaleamplification may contain this region, which can then be cloned usingthe bacterial and yeast markers in the pTEMPhu3 vector.

The pTEMPhu3 cloning vector allows not only selection for homologousrecombinants, but also direct cloning of the integration site in YACS.This vector can also be used to target human chromosome 3, preferablywith a deleted short arm, in a mouse-human monochromosomal microcellhybrid line. Homologous recombinants can be screened by nucleic acidamplification (PCR), and amplification can be screened by DNAhybridization, Southern hybridization, and in situ hybridization. Theamplified region can be cloned into a YAC. This vector and these methodsalso permit a functional analysis of cloned chromosome regions byreintroducing the cloned amplified region into mammalian cells.

B. Preparation of Libraries in YAC Vectors for Cloning of Centromeresand Identification of Functional Chromosomal Units

Another method that may be used to obtain smaller-sized functionalmammalian artificial chromosome units and to clone centromeric DNAinvolves screening of mammalian DNA YAC vector-based libraries andfunctional analysis of potential positive clones in a transgenic mousemodel system. A mammalian DNA library is prepared in a YAC vector, suchas YRT2 [see Schedl et al. (1993) Nuc. Acids Res. 21:4783-4787], whichcontains the murine tyrosinase gene. The library is screened forhybridization to mammalian telomere and centromere sequence probes.Positive clones are isolated and microinjected into pronuclei offertilized oocytes of NMRI/Han mice following standard techniques. Theembryos are then transferred into NMRI/Han foster mothers. Expression ofthe tyrosinase gene in transgenic offspring confers an identifiablephenotype (pigmentation). The clones that give rise totyrosinase-expressing transgenic mice are thus confirmed as containingfunctional mammalian artificial chromosome units.

Alternatively, fragments of SATACs may be introduced into the YACvectors and then introduced into pronuclei of fertilized oocytes ofNMRI/Han mice following standard techniques as above. The clones thatgive rise to tyrosinase-expressing transgenic mice are thus confirmed ascontaining functional mammalian artificial chromosome units,particularly centromeres.

C. Incorporation of Heterologous Genes into Mammalian ArtificialChromosomes through The Use of Homology Targeting Vectors

As described above, the use of mammalian artificial chromosomes forexpression of heterologous genes obviates certain negative effects thatmay result from random integration of heterologous plasmid DNA into therecipient cell genome. An essential feature of the mammalian artificialchromosome that makes it a useful tool in avoiding the negative effectsof random integration is its presence as an extra-genomic gene source inrecipient cells. Accordingly, methods of specific, targetedincorporation of heterologous genes exclusively into the mammalianartificial chromosome, without extraneous random integration into thegenome of recipient cells, are desired for heterologous gene expressionfrom a mammalian artificial chromosome.

One means of achieving site-specific integration of heterologous genesinto artificial chromosomes is through the use of homology targetingvectors. The heterologous gene of interest in subcloned into a targetingvector which contains nucleic acid sequences that are homologous tonucleotides present in the artificial chromosome. The vector is thenintroduced into cells containing the artificial chromosome for specificsite-directed integration into the artificial chromosome through arecombination event at sites of homology between the vector and thechromosome. The homology targeting vectors may also contain selectablemarkers for ease of identifying cells that have incorporated the vectorinto the artificial chromosome as well as lethal selection genes thatare expressed only upon extraneous integration of the vector into therecipient cell genome. Two exemplary homology targeting vectors, λCF-7and pλCF-7-DTA, are described below.

1. Construction of Vector λCF-7

Vector λCF-7 contains the cystic fibrosis transmembrane conductanceregulator [CFTR] gene as an exemplary therapeutic molecule-encodingnucleic acid that may be incorporated into mammalian artificialchromosomes for use in gene therapy applications. This vector, whichalso contains the puromycin-resistance gene as a selectable marker, aswell as the Saccharomyces cerevisiae ura3 gene [orotidine-5-phosphatedecarboxylase], was constructed in a series of steps as follows.

a. Construction of pURA

Plasmid pURA was prepared by ligating a 2.6-kb SalI/XhoI fragment fromthe yeast artificial chromosome vector pYAC5 [Sigma; see also Burke etal. (1987) Science 236:806-812 for a description of YAC vectors as wellas GenBank Accession no. U01086 for the complete sequence of pYAC5]containing the S. cerevisiae ura3 gene with a 3.3-kb SalI/SmaI fragmentof pHyg [see, e.g., U.S. Pat. Nos. 4,997,764, 4,686,186 and 5,162,215,.and the description above]. Prior to ligation the XhoI end was treatedwith Klenow polymerase for blunt end ligation to the SmaI end of the 3.3kb fragment of pHyyg. Thus, pURA contains the S. cerevisiae ura3 gene,and the E. coli ColE1 origin of replication and theampicillin-resistance gene. The uraE gene is included to provide a meansto recover the integrated construct from a mammalian cell as a YACclone.

b. Construction of pUP2

Plasmid pURA was digested with SalI and ligated to a 1.5-kb SalIfragment of pCEPUR. Plasmid pCEPUR is produced by ligating the 1.1 kbSnaBI-NhaI fragment of pBabe-puro [Morgenstern et al. (1990) Nucl. AcidsRes. 18:3587-3596; provided by Dr. L. Székely (Microbiology andTumorbiology Center, Karolinska Institutet, Stockholm); see, alsoTonghua et al. (1995) Chin. Med. J. (Beijing, Engl. Ed.) 108:653-659;Couto et al. (1994) Infect. Immun. 62:2375-2378; Dunckley et al. (1992)FEBS Lett. 296:128-34; French et al. (1995) Anal. Biochem. 228:354-355;Liu et al. (1995) Blood 85:1095-1103; International PCT application Nos.WO 9520044; WO 9500178, and WO 9419456] to the NheI-NruI fragment ofpCEP4 [Invitrogen].

The resulting plasmid, pUP2, contains the all the elements of pURA plusthe puromycin-resistance gene linked to the SV40 promoter andpolyadenylation signal from pCEPUR.

C. Construction of pUP-CFTR

The intermediate plasmid pUP-CFTR was generated in order to combine theelements of pUP2 into a plasmid along with the CFTR gene. First, a4.5-kb SalI fragment of pCMV-CFTR that contains the CFTR-encoding DNA[see, also, Riordan et al. (1989) Science 245:1066-1073, U.S. Pat. No.5,240,846, and Genbank Accession no. M28668 for the sequence of the CFTRgene] containing the CFTR gene only was ligated to XhoI-digested pCEP4[Invitrogen and also described herein] in order to insert the CFTR genein the multiple cloning site of the Epstein Barr virus-based (EBV)vector pCEP4 [Invitrogen, San Diego, Calif.; see also Yates et al.(1985) Nature 313:812-815; see, also U.S. Pat. No. 5,468,615] betweenthe CMV promoter and SV40 polyadenylation signal. The resulting plasmidwas designated pCEP-CFTR. Plasmid pCEP-CFTR was then digested with SalIand the 5.8-kb fragment containing the CFTR gene flanked by the CMVpromoter and SV40 polyadenylation signal was ligated to SalI-digestedpUP2 to generate pUP-CFTR. Thus, pUP-CFTR contains all elements of pUP2plus the CFTR gene linked to the CMV promoter and SV40 polyadenylationsignal.

d. Construction of λCF-7

Plasmid pUP-CFTR was then linearized by partial digestion with EcoRI andthe 13 kb fragment containing the CFTR gene was ligated withEcoRI-digested Charon 4Aλ [see Blattner et al. (1977) Science 196:161;Williams and Blattner (1979) J. Virol. 29:555 and Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring HarborLaboratory Press, Volume 1, Section 2.18, for descriptions of Charon4Aλ]. The resulting vector, λCF8, contains the Charon 4Aλ bacteriophageleft arm, the CFTR gene linked to the CMV promoter and SV40polyadenylation signal, the ura3 gene, the puromycin-resistance genelinked to the SV40 promoter and polyadenylation signal, the thymidinekinase promoter [TK], the ColE1 origin of replicaton, the amplicillanresistance gene and the Charon 4Aλ bacteriophage right arm. The λCF8construct was then digested with XhoI and the resulting 27.1 kb wasligated to the 0.4kb XhoI/EcoRI fragment of pJBP86 [described below],containing the SV40 polyA signal and the EcoRI-digested Charon 4A λright arm. The resulting vector λCF-7 contains the Charon 4Aλ left arm,the CFTR encoding DNA linked to the CMV promoter and SV40 polyA signal,the ura3 gene, the puromycin resistance gene linked to the SV40 promoterand polyA signal and the Charon 4λ right arm. The λ DNA fragmentsprovide encode sequences homologous to nucleotides present in theexemplary artificial chromosomes.

The vector is then introduced into cells containing the artificialchromosomes exemplified herein. Accordingly, when the linear λCF-7vector is introduced into megachromosome-carrying fusion cell lines,such as described herein, it will be specifically integrated into themegachromosome through recombination between the homologousbacteriophage λ sequences of the vector and the artificial chromosome.

2. Construction of Vector λCF-7-DTA

Vector λCF-7-DTA also contains all the elements contained in λCF-7, butadditionally contains a lethal selection marker, the diptheria toxin-A(DT-A) gene as well as the ampicillin-resistance gene and an origin ofreplication. This vector was constructed in a series of steps asfollows.

a. Construction of pJBP86

Plasmid pJBP86 was used in the construction of λCF-7, above. A 1.5-kbSalI fragment of pCEPUR containing the puromycin-resistance gene linkedto the SV40 promoter and polyadenylation signal was ligated toHindIII-digested pJB8 [see, e.g., Ish-Horowitz et al. (1981) NucleicAcids Res. 9:2989-2998; available from ATCC as Accession No. 37074;commercially available from Amersham, Arlington Heights, Ill.]. Prior toligation the SalI ends of the 1.5 kb fragment of pCEPUR and th4 HindIIIlinearized pJB8 ends were treated with Klenow polymerase. The resultingvector pJBP86 contains the puromycin resistance gene linked to the SV40promoter and polyA signal, the 1.8 kb COS region of Charon 4Aλ, theColE1 origin of replication and the ampicillin resistance gene.

b. Construction of pMEP-DTA

A 1.1-kb XhoI/SalI fragment of pMC1-DT-A [see, e.g., Maxwell et al.(1986) Cancer Res. 46:4660-4666] containing the diptheria toxin-A genewas ligated to XhoI-digested pMEP4 [Invitrogen, San Diego, Calif.] togenerate pMEP-DTA. To produce pMC1-DT-A, the coding region of the DTAgene was isolated as a 800 bp PstIHindIII fragment from p2249-1 andinserted into pMC1neopolyA [pMC1 available from Stratagene] in place ofthe neo gene and under the control of the TK promotoer. The resultingconstruct pMC1DT-A was digested with HindIII, the ends filled by Klenowand SalI linkers were ligated to produce a 1061 bp TK-DTA gene cassettewith an XhoI end [5′] and a SalI end containing the 270 bp TK promoterand the ˜790 bp DT-A fragment. This fragment was ligated intoXhoI-digested pMEP4 .

Plasmid pMEP-DTA thus contains the DT-A gene linked to the TK promoterand SV40, ColE1 origin of replication and the ampicillin-resistancegene.

c. Construction of pJB83-DTA9

Plasmid pJB8 was digested with HindIII and ClaI and ligated with anoligonucleotide [see SEQ ID NOs. 7 and 8 for the sense and antisensestrands of the oligonucleotide, respectively] to generate pJB83. Theoligonucleotide that was ligated to ClaI/HindIII-digested pJB8 containedthe recognition sites of SwaI, PacI and SrfI restriction endonucleases.These sites will permit ready linearization of the pλCF-7-DTA construct.

Next, a 1.4-kb XhoI/SalI fragment of pMEP-DTA, containing the DT-A genewas ligated to SalI-digested pJB83 to generate pJB83-DTA9.

d. Construction of λCF-7-DTA

The 12-bp overhangs of λCF-7 were removed by Mung bean nuclease andsubsequent T4 polymerase treatments. The resulting 41.1-kb linear λCF-7vector was then ligated to pFB83-DTA9 which had been digested with ClaIand treated with T4 polymerase. The resulting vector, λCF-7-DTA,contains all the elements of λCF-7 as well as the DT-A gene linked tothe TK promoter and the SV40 polyadenylation signal, the 1.8 kB Charon4Aλ COS region, the ampicillin-resistance gene[from pJB83-DTA9] and theCol E1 origin of replication [from pJB83-DT9A].

D. Targeting Vectors Using Luciferase Markers: Plasmid pMCT-RUC

Plasmid pMCT-RUC [14 kbp] was constructed for site-specific targeting ofthe Renilla luciferase [see, e.g., U.S. Pat. Nos. 5,292,658 and5,418,155 for a description of DNA encoding Renilla luciferase, andplasmid pTZrLuc-1, which can provide the starting material forconstruction of such vectors] gene to a mammalian artificial chromosome.The relevant features of this plasmid are the Renilla luciferase geneunder transcriptional control of the human cytomegalovirusimmediate-early gene enhancer/promoter; the hygromycin-resistance genea, positive selectable marker, under the transcriptional control of thethymidine kinase promoter. In particular, this plasmid contains plasmidpAG60 [see, e.g., U.S. Pat. Nos. 5,118,620, 5,021,344, 5,063,162 and4,946,952; see, also Colbert-Garapin et al. (1981) J. Mol. Biol.150:1-14], which includes DNA (i.e., the neomycin-resistance gene)homologous to the minichromosome, as well as the Renilla andhygromycin-resistance genes, the HSV-tk gene under control of the tkpromoter as a negative selectable marker for homologous recombination,and a unique HpaI site for linearizing the plasmid.

This construct was introduced, via calcium phosphate transfection, intoEC3/7C5 cells [see, Lorenz et al. (1996) J. Biolum. Chemilum. 11:31-37].The EC3/7C5 cells were maintained as a monolayer [see, Gluzman (1981)Cell 23:175-183]. Cells at 50% confluency in 100 mm Petri dishes wereused for calcium phosphate transfection [see, Harper et al. (1981)Chromosoma 83:431-4391 using 10 μg of linearized pMCT-RUC per plate.Colonies originating from single transfected cells were isolated andmaintained in F-12 medium containing hygromycin (300 μg/mL) and 10%fetal bovine serum. Cells were grown in 100 mm Petri dishes prior to theRenilla luciferase assay.

The Renilla luciferase assay was performed [see, e.g., Matthews et al.(1977) Biochemistry 16:85-91]. Hygromycin-resistant cell lines obtainedafter transfection of EC3/7C5 cells with linearized plasmid pMCT-RUC[“B” cell lines] were grown to 100% confluency for measurements of lightemission in vivo and in vitro. Light emission was measured in vivo afterabout 30 generations as follows: growth medium was removed and replacedby 1 mL RPMI 1640 containing coelenterazine [1 mmol/L finalconcentration]. Light emission from cells was then visualized by placingthe Petri dishes in a low light video image analyzer [HamamatsuArgus-1001. An image was formed after 5 min. of photon accumulationusing 100% sensitivity of the photon counting tube. For measuring lightemission in vitro, cells were trypsinized and harvested from one Petridish, pelleted, resuspended in 1 mL assay buffer [0.5 mol/L NaCl, 1mmol/L EDTA, 0.1 mol/L potassium phosphate, pH 7.4] and sonicated on icefor 10 s. Lysates were than assayed in a Turner TD-20e luminometer for10 s after rapid injection of 0.5 mL of 1 mmol/L coelenterazine, and theaverage value of light emission was recorded as LU [1 LU=1.6×106 hu/sfor this instrument].

Independent cell lines of EC3/7C5 cells transfected with linearizedplasmid pMCT-RUC showed different levels of Renilla luciferase activity.Similar differences in light emission were observed when measurementswere performed on lysates of the same cell lines. This variation inlight emission was probably due to a position effect resulting from therandom integration of plasmid pMCT-RUC into the mouse genome, sinceenrichment for site targeting of the luciferase gene was not performedin this experiment.

To obtain transfectant populations enriched in cells in which theluciferase gene had integrated into the minichromosome, transfectedcells were grown in the presence of ganciclovis. This negative selectionmedium selects against cells in which the added pMCT-RUC plasmidintegrated into the host EC3/7C5 genome. This selection thereby enrichesthe surviving transfectant population with cells containing pMCT-RUC inthe minichromosome. The cells surviving this selection were evaluated inluciferase assays which revealed a more uniform level of luciferaseexpression. Additionally, the results of in situ hybridization assaysindicated that the Renilla luciferase gene was contained in theminichromosome in these cells, which further indicates successfultargeting of pMCT-RUC into the minichromosome.

Plasmid pNEM-1, a variant of pMCT-RUC which also contains λ DNA toprovide an extended region of homology to the minichromosome [see, othertargeting vectors, below], was also used to transfect EC3/7C5 cells.Site-directed targeting of the Renilla luciferase gene and thehygromycin-resistance gene in pNEM-1 to the minichromosome in therecipient EC3/7C5 cells was achieved. This was verified by DNAamplification analysis and by in situ hybridization. Additionally,luciferase gene expression was confirmed in luciferase assays of thetransfectants.

E. Protein Secretion Targeting Vectors

Isolation of heterologous proteins produced intracellularly in mammaliancell expression systems requires cell disruption under potentially harshconditions and purification of the recombinant protein from cellularcontaminants. The process of protein isolation may be greatlyfacilitated by secretion of the recombinantly produced protein into theextracellular medium where there are fewer contaminants to remove duringpurification. Therefore, secretion targeting vectors have beenconstructed for use with the mammalian artificial chromosome system.

A useful model vector for demonstrating production and secretion ofheterologous protein in mammalian cells contains DNA encoding a readilydetectable reporter protein fused to an efficient secretion signal thatdirects transport of the protein to the cell membrane and secretion ofthe protein from the cell. Vectors pLNCX-ILRUC and pLNCX-ILRUCλ,described below, are examples of such vectors. These vectors contain DNAencoding an interleukin-2 (IL2) signal peptide-Renilla reniformisluciferase fusion protein. The IL-2 signal peptide [encoded by thesequence set forth in SEQ ID No. 9] directs secretion of the luciferaseprotein, to which it is linked, from mammalian cells. Upon secretionfrom the host mammalian cell, the IL-2 signal peptide is cleaved fromthe fusion protein to deliver mature, active, luciferase protein to theextracellular medium. Successful production and secretion of thisheterologous protein can be readily detected by performing luciferaseassays which measure the light emitted upon exposure of the medium tothe bioluminescent luciferin substrate of the luciferase enzyme. Thus,this feature will be useful when artificial chromosomes are used forgene therapy. The presence of a functional artificial chromosomecarrying an IL-Ruc fusion with the accompanying therapeutic genes willbe readily monitored. Body fluids or tissues can be sampled and testedfor luciferase expression by adding luciferin and appropriate cofactorsand observing the bioluminescence.

1. Construction of Protein Secretion Vector pLNCX-ILRUC

Vector pLNCX-ILRUC contains a human IL-2 signal peptide-R. reniformisfusion gene linked to the human cytomegalovirus (CMV) immediate earlypromoter for constitutive expression of the gene in mammalian cells. Theconstruct was prepared as follows.

a. Preparation of the IL-2 Signal Sequence-Encoding DNA

A 69-bp DNA fragment containing DNA encoding the human IL-2 signalpeptide was obtained through nucleic acid amplification, usingappropriate primers for IL-2, of an HEK 293 cell line [see, e.g., U.S.Pat. No. 4,518,584 for an IL-2 encoding DNA; see, also SEQ ID No. 9; theIL-2 gene and corresponding amino acid sequence is also provided in theGenbank Sequence Database as accession nos. K02056 and J002641. Thesignal peptide includes the first 20 amino acids shown in thetranslations provided in both of these Genbank entries and in SEQ ID NO.9. The corresponding nucleotide sequence encoding the first 20 aminoacids is also provided in these entries [see, e.g., nucleotides 293-52of accession no. K02056 and nucleotides 478-537 of accession no.J00264), as well as in SEQ ID NO. 9. The amplification primers includedan EcoRI site [GAATTC] for subcloning of the DNA fragment after ligationinto pGEMT [Promega]. The forward primer is set forth in SEQ ID No. 11and the sequence of the reverse primer is set forth in SEQ ID No. 12.forward [SEQ ID No. 11] TTTGAATTCATGTACAGGATGCAACTCCTG reverse [SEQ IDNo. 12] TTTGAATTCAGTAGGTGCACTGTTTGTGAC

b. Preparation of the R. reniformis Luciferase-Encoding DNA

The initial source of the R. reniformis luciferase gene was plasmidpLXSN-RUC. Vector pLXSN [see, e.g., U.S. Pat. Nos. 5,324,655, 5,470,730,5,468,634, 5,358,866 and Miller et al. (1989) Biotechniques 7:980] is aretroviral vector capable of expressing heterologous DNA under thetranscriptional control of the retroviral LTR; it also contains theneomycin-resistance gene operatively linked for expression to the SV40early region promoter. The R. reniformis luciferase gene was obtainedfrom plasmid pTZrLuc-1 [see, e.g., U.S. Pat. No. 5,292,658; see also theGenbank Sequence Database accession no. M63501; and see also Lorenz etal. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:4438-4442] and is shown asSEQ ID NO. 10. The 0.97 kb EcoRI/SmaI fragment of pTZrLuc-1 contains thecoding region of the Renilla luciferase-encodig DNA. Vector pLXSN wasdigested with and ligated with the luciferase gene contained on apLXSN-RUC, which contains the luciferase gene located operably linked tothe viral LTR and upstream of the SV40 promoter, which directsexpression of the neomycin-resistance gene.

c. Fusion of DNA Encoding the IL-2 Signal Peptide and the R. reniformisLuciferase Gene to Yield pLXSN-ILRUC

The pGEMT vector containing the IL-2 signal peptide-encoding DNAdescribed in 1.a. above was digested with EcoRI, and the resultingfragment encoding the signal peptide was ligated to EcoRI-digestedpLXSN-RUC. The resulting plasmid, called pLXSN-ILRUC, contains the IL-2signal peptide-encoding DNA located immediately upstream of the R.reniformis gene in pLXSN-RUC. Plasmid pLXSN-ILRUC was then used as atemplate for nucleic acid amplification of the fusion gene in order toadd a SmaI site at the 3′ end of the fusion gene. The amplificationproduct was subcloned into linearized [EcoRI/SmaI-digested] pGEMT[Promega] to generate ILRUC-pGEMT.

d. Introduction of the Fusion Gene into a Vector Containing ControlElements for Expression in Mammalian Cells

Plasmid ILRUC-pGEMT was digested with KspI and SmaI to release afragment containing the IL-2 signal peptide-luciferase fusion gene whichwas ligated to HpaI-digested pLNCX. Vector pLNCX [see, e.g., U.S. Pat.Nos. 5,324,655 and 5,457,182; see, also Miller and Rosman (1989)Biotechniques 7:980-9901 is a retroviral vector for expressingheterologous DNA under the control of the CMV promoter; it also containsthe neomycin-resistance gene under the transcriptional control of aviral promoter. The vector resulting from the ligation reaction wasdesignated pLNCX-ILRUC. Vector pLNCX-ILRUC contains the IL-2 signalpeptide-luciferase fusion gene located immediately downstream of the CMVpromoter and upstream of the viral 3′ LTR and polyadenylation signal inpLNCX. This arrangement provides for expression of the fusion gene underthe control of the CMV promoter. Placement of the heterologousprotein-encoding DNA [i.e., the luciferase gene] in operative linkagewith the IL-2 signal peptide-encoding DNA provides for expression of thefusion in mammalian cells transfected with the vector such that theheterologous protein is secreted from the host cell into theextracellular medium.

2. Construction of Protein Secretion Targeting Vector pLNCX-ILRUCλ

Vector pLNCX-ILRUC may be modified so that it can be used to introducethe IL-2 signal peptide-luciferase fusion gene into a mammalianartificial chromosome in a host cell. To facilitate specificincorporation of the pLNCX-ILRUC expression vector into a mammalianartificial chromosome, nucleic acid sequences that are homologous tonucleotides present in the artificial chromosome are added to the vectorto permit site directed recombination.

Exemplary artificial chromosomes described herein contain λ phage DNA.Therefore, protein secretion targeting vector pLNCX-ILRUCλ was preparedby addition of λ phage DNA [from Charon 4A arms] to produce thesecretion vector pLNCX-ILRUC.

3. Expression and Secretion of R. reniformis Luciferase from MammalianCells

a. Expression of R. reniformis Luciferase Using pLNCX-ILRUC

Mammalian cells [LMTK⁻ from the ATCC] were transiently transfected withvector pLNCX-ILRUC [˜10 μg] by electroporation [BIORAD, performedaccording to the manufacturer's instructions]. Stable transfectantsproduced by growth in G418 for neo selection have also been prepared.

Transfectants were grown and then analyzed for expression of luciferase.To determine whether active luciferase was secreted from the transfectedcells, culture media were assayed for luciferase by addition ofcoelentrazine [see, e.g., Matthews et al. (1977) Biochemistry 16:85-91].

The results of these assays establish that vector pLNCX-ILRUC is capableof providing constitutive expression of heterologous DNA in mammalianhost cells. Furthermore, the results demonstrate that the human IL-2signal peptide is capable of directing secretion of proteins fused tothe C-terminus of the peptide. Additionally, these data demonstrate thatthe R. reniformis luciferase protein is a highly effective reportermolecule, which is stable in a mammalian cell environment, and forms thebasis of a sensitive, facile assay for gene expression.

b. Renilla reniformis Luciferase Appears to be Secreted from LMTK⁻Cells.

(i) Renilla Luciferase Assay of Cell Pellets

The following cells were tested:

-   -   cells with no vector: LMTK⁻ cells without vector as a negative        control;    -   cells transfected with pLNCX only;    -   cells transfected with RUC-pLNCX [Renilla luciferase gene in        pLNCX vector];    -   cells transfected with pLNCX-ILRUC [vector containing the IL-2        leader sequence+Renilla luciferase fusion gene in pLNCX vector].

Forty-eight hours after electroporation, the cells and culture mediumwere collected. The cell pellet from 4 plates of cells was resuspendedin 1 ml assay buffer and was lysed by sonication. Two hundred μl of theresuspended cell pellet was used for each assay for luciferase activity[see, e.g., Matthews et al. (1977) Biochemistry 16:85-91]. The assay wasrepeated three times and the average bioluminescence measurement wasobtained.

The results showed that there was relatively low backgroundbioluminescence in the cells transformed with pLNCX or the negativecontrol cells; there was a low level observed in the cell pellet fromcells containing the vector with the IL-2 leader sequence-luciferasegene fusion and more than 5000 RLU in the sample from cells containingRUC-pLNCX.

(ii) Renilla Luciferase Assay of Cell Medium

Forty milliliters of medium from 4 plates of cells were harvested andspun down. Two hundred microliters of medium was used for eachluciferase activity assay. The assay was repeated several times and theaverage bioluminescence measurement was obtained. These results showedthat a relatively high level of bioluminescence was detected in the cellmedium from cells transformed with pLNCX-ILRUC; about 10-fold lowerlevels [slightly above the background levels in medium from cells withno vector or transfected with pLNCX only] was detected in the cellstransfected with RUC-pLNCX.

(iii) Conclusions

The results of these experiments demonstrated that Renilla luciferaseappears to be secreted from LMTK⁻ cells under the direction of the IL-2signal peptide. The medium from cells transfected with Renillaluciferase-encoding DNA linked to the DNA encoding the IL-2 secretionsignal had substantially higher levels of Renilla luciferase activitythan controls or cells containing luciferase-encoding DNA without thesignal peptide-encoding DNA. Also, the differences between the controlsand cells containing luciferase encoding-DNA demonstrate that theluciferase activity is specifically from luciferase, not from anon-specific reaction. In addition, the results from the medium ofRUC-pLNCX transfected cells, which is similar to background, show thatthe luciferase activity in the medium does not come from cell lysis, butfrom secreted luciferase.

c. Expression of R. reniformis Luciferase Using pLNCX-ILRUCλ

To express the IL-2 signal peptide-R. reniformis fusion gene from anmammalian artificial chromosome, vector pLNCX-ILRUCλ is targeted forsite-specific integration into a mammalian artificial chromosome throughhomologous recombination of the λ DNA sequences contained in thechromosome and the vector. This is accomplished by introduction ofpLNCX-ILRUCλ into either a fusion cell line harboring mammalianartificial chromosomes or mammalian host cells that contain mammalianartificial chromosomes. If the vector is introduced into a fusion cellline harboring the artificial chromosomes, for example throughmicroinjection of the vector or transfection of the fusion cell linewith the vector, the cells are then grown under selective conditions.The artificial chromosomes, which have incorporated vector pLNCX-ILRUCλ,are isolated from the surviving cells, using purification procedures asdescribed above, and then injected into the mammalian host cells.

Alternatively, the mammalian host cells may first be injected withmammalian artificial chromosomes which have been isolated from a fusioncell line. The host cells are then transfected with vector pLNCX-ILRUCλand grown.

The recombinant host cells are then assayed for luciferase expression asdescribed above.

F. Other Targeting Vectors

These vectors, which are based on vector pMCT-RUC, rely on positive andnegative selection to insure insertion and selection for the doublerecombinants. A single crossover results in incorporation of the DT-A,which kills the cell, double crossover recombinations delete the DT-1gene.

1. Plasmid pNEM1 contains:

-   -   DT-A: Diphtheria toxin gene (negative selectable marker)    -   Hyg: Hygromycin gene (positive selectable marker)    -   ruc: Renilla luciferase gene (non-selectable marker)    -   1: LTR-MMTV promoter    -   2: TK promoter    -   3: CMV promoter    -   MMR: Homology region (plasmid pAG60)

2. Plasmid pNEM-2 and -3 are similar to pNEM 1 except for differentnegative selectable markers:

-   -   pNEM-1: diphtheria toxin gene as “-” selectable marker    -   pNEM-2: hygromycin antisense gene as “-” selectable marker    -   pNEM-3: thymidine kinase HSV-1 gene as “-” selectable marker

3. Plasmid—λ DNA based homology:

-   -   pNEMλ-1: base vector    -   pNEMλ-2: base vector containing p5=gene    -   1: LTR MMTV promoter    -   2: SV40 promoter    -   3: CMV promoter    -   4: μTIIA promoter (metallothionein gene promoter)—homology        region (plasmid pAG60)    -   λ L.A. and λ R.A. homology regions for λ left and right arms (λ        gt-WES).

EXAMPLE 13

Microinjection of Mammalian Cells with Plasmid DNA

These procedures will be used to microinject MACs into eukaryotic cells,including mammalian and insect cells.

The microinjection technique is based on the use of small glasscapillaries as a delivery system into cells and has been used forintroduction of DNA fragments into nuclei [see, e.g., Chalfie et al.(1994) Science 263:802-804]. It allows the transfer of almost any typeof molecules, e.g., hormones, proteins, DNA and RNA, into either thecytoplasm or nuclei of recipient cells This technique has no cell typerestriction and is more efficient than other methods, includingCa²⁺-mediated gene transfer and liposome-mediated gene transfer. About20-30% of the injected cells become successfully transformed.

Microinjection is performed under a phase-contrast microscope. A glassmicrocapillary, prefilled with the DNA sample, is directed into a cellto be injected with the aid of a micromanipulator. An appropriate samplevolume (1-10 pl) is transferred into the cell by gentle air pressureexerted by a transjector connected to the capillary. Recipient cells aregrown on glass slides imprinted with numbered squares for convenientlocalization of the injected cells.

a. Materials and Equipment

Nunclon tissue culture dishes 35×10 mm, mouse cell line EC3/7C5 PlasmidDNA pCH110 [Pharmacia], Purified Green Florescent Protein (GFP) [GFPsfrom Aequorea and Renilla have been purified and also DNA encoding GFPshas been cloned; see, e.g., Prasher et al. (1992) Gene 111:229-233;International PCT Application No. WO 95/07463, which is based on U.S.application Ser. No. 08/119,678 and U.S. application Ser. No.08/192,2741, ZEISS Axiovert 100 microscope, Eppendorf transjector 5246,Eppendorf micromanipulator 5171, Eppendorf Cellocate coverslips,Eppendorf microloaders, Eppendorf femtotips and other standardequipment.

b. Protocol for Injecting

(1) Fibroblast cells are grown in 35 mm tissue culture dishes (37° C.,5% CO₂) until the cell density reaches 80% confluency. The dishes areremoved from the incubator and medium is added to about a 5 mm depth.

(2) The dish is placed onto the dish holder and the cells observed with10 × objective; the focus is desirably above the cell surface.

(3) Plasmid or chromosomal DNA solution [1 ng/μl] and GFP proteinsolution are further purified by centrifuging the DNA sample at a forcesufficient to remove any particular debris [typically about 10,000 rpmfor 10 minutes in a microcentrifuge].

(4) Two 2 μl of the DNA solution (1 ng/μl) is loaded into amicrocapillary with an Eppendorf microloader. During loading, the loaderis inserted to the tip end of the microcapillary. GFP (1 mg/ml) isloaded with the same procedure.

(5) The protecting sheath is removed from the microcapillary and themicrocapillary is fixed onto the capillary holder connected with themicromanipulator.

(6) The capillary tip is lowered to the surface of the medium and isfocussed on the cells gradually until the tip of the capillary reachesthe surface of a cell. The capillary is lowered further so that the itis inserted into the cell. Various parameters, such as the level of thecapillary, the time and pressure, are determined for the particularequipment. For example, using the fibroblast cell line C5 and theabove-noted equipment, the best conditions are: injection time 0.4second, pressure 80 psi. DNA can then be automatically injected into thenuclei of the cells.

(7) After injection, the cells are returned to the incubator, andincubated for about 18-24 hours.

(8) After incubation the number of transformants can be determined by asuitable method, which depends upon the selection marker. For example,if green fluorescent protein is used, the assay can be performed usingUV light source and fluorescent filter set at 0-24 hours afterinjection. If β-gal-containing DNA, such as DNA-derived from pHC110, hasbeen injected, then the transformants can be assayed for β-gal.

(c) Detection of β-galactosidase in Cells Injected with Plasmid DNA

The medium is removed from the culture plate and the cells are fixed byaddition of 5 ml of fixation Solution I: (1% glutaraldehyde; 0.1 Msodium phosphate buffer, pH 7.0; 1 mM MgCl₂), and incubated for 15minutes at 37° C. Fixation Solution I is replaced with 5 ml of X-galSolution II: [0.2% X-gal, 10 mM sodium phosphate buffer (pH 7.0), 150 mMNaCl, 1 mM MgCl₂, 3.3 mM K₄Fe(CN)₆H₂O, 3.3 mM K₃Fe(CN)₆], and the platesare incubated for 30-60 minutes at 37° C. The X-gal solution is removedand 2 ml of 70% glycerol is added to each dish. Blue stained cells areidentified under a light microscope.

This method will be used to introduce a MAC, particularly the MAC withthe anti-HIV megachromosome, to produce a mouse model for anti-HIVactivity.

EXAMPLE 14

Transgenic (Non-Human) Animals

Transgenic (non-human) animals can be generated that expressheterologous genes which confer desired traits, e.g., diseaseresistance, in the animals. A transgenic mouse is prepared to serve as amodel of a disease-resistant animal. Genes that encode vaccines or thatencode therapeutic molecules can be introduced into embryos or ES cellsto produce animals that express the gene product and thereby areresistant to or less susceptible to a particular disorder.

The mammalian artificial megachromosome and others of the artificialchromosomes, particularly the SATACs, can be used to generate transgenic(non-human) animals, including mammals and birds, that stably expressgenes conferring desired traits, such as genes conferring resistance topathogenic viruses. The artificial chromosomes can also be used toproduce transgenic (non-human) animals, such as pigs, that can produceimmunologically humanized organs for xenotransplantation.

For example, transgenic mice containing a transgene encoding an anti-HIVribozyme provide a useful model for the development of stable transgenic(non-human) animals using these methods. The artificial chromosomes canbe used to produce transgenic (non-human) animals, particularly, cows,goats, mice, oxen, camels, pigs and sheep, that produce the proteins ofinterest in their milk; and to produce transgenic chickens and otheregg-producing fowl, that produce therapeutic proteins or other proteinsof interest in their eggs. For example, use of mammary gland-specificpromoters for expression of heterologous DNA in milk is known [see, e.g.U.S. Pat. No. 4,873,316]. In particular, a milk-specific promoter or apromoter, preferably linked to a milk-specific signal peptide,specifically activated in mammary tissue is operatively linked to theDNA of interest, thereby providing expression of that DNA sequence inmilk.

1. Development of Control Transgenic Mice Expressing Anti-HIV Ribozyme

Control transgenic mice are generated in order to compare stability andamounts of transgene expression in mice developed using transgene DNAcarried on a vector (control mice) with expression in mice developedusing transgenes carried in an artificial megachromosome.

a. Development of Control Transgenic Mice Expressing β-galactosidase

One set of control transgenic mice was generated by microinjection ofmouse embryos with the β-galactosidase gene alone. The microinjectionprocedure used to introduce the plasmid DNA into the mouse embryos is asdescribed in Example 13, but modified for use with embryos [see, e.g.,Hogan et al. (1994) Manipulating the Mouse Embryo, A: Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., see,especially pages 255-264 and Appendix 3]. Fertilized mouse embryos[Strain CB6 obtained from Charles River Co.] were injected with 1 ng ofplasmid pCH110 (Pharmacia) which had been linearized by digestion withBamHI. This plasmid contains the β-galactosidase gene linked to the SV40late promoter. The β-galactosidase gene product provides a readilydetectable marker for successful transgene expression. Furthermore,these control mice provide confirmation of the microinjection procedureused to introduce the plasmid into the embryos. Additionally, becausethe megachromosome that is transferred to the mouse embryos in the modelsystem (see below) also contains the β-galactosidase gene, the controltransgenic mice that have been generated by injection of pCH110 intoembryos serve as an analogous system for comparison of heterologous geneexpression from a plasmid versus from a gene carried on an artificalchromosome.

After injection, the embryos are cultured in modified HTF medium under5% CO₂ at 37° C. for one day until they divide to form two cells. Thetwo-cell embryos are then implanted into surrogate mother female mice[for procedures see, Manipulating the Mouse Embryo, A Laboratory Manual(1994) Hogan et al., eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., pp. 127 et seq.].

b. Development of Control Transgenic Mice Expressing Anti-HIV Ribozyme

One set of anti-HIV ribozyme gene-containing control transgenic mice wasgenerated by microinjection of mouse embryos with plasmid pCEPUR-132which contains three different genes: (1) DNA encoding an anti-HIVribozyme, (2) the puromycin-resistance gene and (3) thehygromycin-resistance gene. Plasmid pCEPUR-132 was constructed byligating portions of plasmid pCEP-132 containing the anti-HIV ribozymegene (referred to as ribozyme D by Chang et al. [(1990) Clin. Biotech.2:23-31]; see also U.S. Pat. No. 5,144,019 to Rossi et al., particularlyFIG. 4 of the patent) and the hygromycin-resistance gene with a portionof plasmid pCEPUR containing the puromycin-resistance gene.

Plasmid pCEP-132 was constructed as follows. Vector pCEP4 (Invitrogen,San Diego, Calif.; see also Yates et al. (1985) Nature 313:812-815) wasdigested with XhoI which cleaves in the multiple cloning site region ofthe vector. This ˜10.4-kb vector contains the hygromycin-resistance genelinked to the thymidine kinase gene promoter and polyadenylation signal,as well as the ampicillin-resistance gene and ColE1 origin ofreplication and EBNA-1 (Epstein-Barr virus nuclear antigen) genes andOriP. The multiple cloning site is flanked by the cytomegaloviruspromoter and SV40 polyadenylation signal.

XhoI-digested pCEP4 was ligated with a fragment obtained by digestion ofplasmid 132 (see Example 4 for a description of this plasmid) with XhoIand SalI. This XhoI/SalI fragment contains the anti-HIV ribozyme genelinked at the 3′ end to the SV40 polyadenylation signal. The plasmidresulting from this ligation was designated pCEP-132. Thus, in effect,pCEP-132 comprises pCEP4 with the anti-HIV ribozyme gene and SV40polyadenylation signal inserted in the multiple cloning site for CMVpromoter-driven expression of the anti-HIV ribozyme gene.

To generate pCEPUR-132, pCEP-132 was ligated with a fragment of pCEPUR.pCEPUR was prepared by ligating a 7.7-kb fragment generated uponNheI/NruI digestion of pCEP4 with a 1.1-kb NheI/SnaBI fragment of pBabe[see Morgenstern and Land (1990) Nucleic Acids Res. 18:3587-3596 for adescription of pBabe] that contains the puromycin-resistance gene linkedat the 5′ end to the SV40 promoter. Thus, pCEPUR is made up of theampicillin-resistance and EBNA1 genes, as well as the ColE1 and OriPelements from pCEP4 and the puromycin-resistance gene from pBabe. Thepuromycin-resistance gene in pCEPUR is flanked by the SV40 promoter(from pBabe) at the 5′ end and the SV40 polyadenylation signal (frompCEP4) at the 3′ end.

Plasmid pCEPUR was digested with XhoI and SalI and the fragmentcontaining the puromycin-resistance gene linked at the 5′ end to theSV40 promoter was ligated with XhoI-digested pCEP-132 to yield the˜12.1-kb plasmid designated pCEPUR-132. Thus, pCEPUR-132, in effect,comprises pCEP-132 with puromycin-resistance gene and SV40 promoterinserted at the XhoI site. The main elements of pCEPUR-132 are thehygromycin-resistance gene linked to the thymidine kinase promoter andpolyadenylation signal, the anti-HIV ribozyme gene linked to the CMVpromoter and SV40 polyadenylation signal, and the puromycin-resistancegene linked to the SV40 promoter and polyadenylation signal. The plasmidalso contains the ampicillin-resistance and EBNA1 genes and the ColE1origin of replication and OriP.

Zygotes were prepared from (C57BL/6J×CBA/J) F1 female mice [see, e.g.,Manipulating the Mouse Embryo, A Laboratory Manual (1994) Hogan et al.,eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., p.429], which had been previously mated with a (C57BL/6J×CBA/J) F1 male.The male pronuclei of these F2 zygotes were injected [see, Manipulatingthe Mouse Embryo, A Laboratory Manual (1994) Hogan et al., eds., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.] withpCEPUR-132 (˜3 μg/ml), which had been linearized by digestion with NruI.The injected eggs were then implanted in surrogate mother female micefor development into transgenic offspring.

These primary carrier offspring were analyzed (as described below) forthe presence of the transgene in DNA isolated from tail cells. Sevencarrier mice that contained transgenes in their tail cells (but that maynot carry the transgene in all their cells, i.e., they may be chimeric)were allowed to mate to produce non-chimeric or germ-line heterozygotes.The heterozygotes were, in turn, crossed to generate homozygotetransgenic offspring.

2. Development of Model Transgenic Mice Using Mammalian ArtificialChromosomes

Fertilized mouse embryos are microinjected (as described above) withmegachromosomes (1-10 pL containing 0-1 chromosomes/pL) isolated fromfusion cell line G3D5 or H1D3 (described above). The megachromosomes areisolated as described herein. Megachromosomes isolated from either cellline carry the anti-HIV ribozyme (ribozyme D) gene as well as thehygromycin-resistance and β-galactosidase genes. The injected embryosare then developed into transgenic mice as described above.

Alternatively, the megachromosome-containing cell line G3D5* or H1D3* isfused with mouse embryonic stem cells [see, e.g., U.S. Pat. No.5,453,357, commerically available; see Manipulating the Mouse Embryo, ALaboratory Manual (1994) Hogan et al., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pages 253-289] followingstandard procedures see also, e.g., Guide to Techniques in MouseDevelopment in Methods in Enzymology Vol. 25, Wassarman and DePamphilis, eds. (1993), pages 803-932]. (It is also possible to deliverisolated megachromosomes into embryonic stem cells using the Microcellprocedure [such as that described above].) The stem cells are culturedin the presence of a fibroblast [e.g., STO fibroblasts that areresistant to hygromycin and puromycin]. Cells of the resultant fusioncell line, which contains megachromosomes carrying the transgenes [i.e.,anti-HIV ribozyme, hygromycin-resistance and β-galactosidase genes], arethen transplanted into mouse blastocysts, which are in turn implantedinto a surrogate mother female mouse where development into a transgenicmouse will occur.

Mice generated by this method are chimeric; the transgenes will beexpressed in only certain areas of the mouse, e.g., the head, and thusmay not be expressed in all cells.

3. Analysis of Transgenic Mice for Transgene Expression

Beginning when the transgenic mice, generated as described above, arethree-to-four weeks old, they can be analyzed for stable expression ofthe transgenes that were transferred into the embryos [or fertilizedeggs] from which they develop. The transgenic mice may be analyzed inseveral ways as follows.

a. Analysis of Cells Obtained from the Transgenic Mice

Cell samples [e.g., spleen, liver and kidney cells, lymphocytes, tailcells] are obtained from the transgenic mice. Any cells may be testedfor transgene expression. If, however, the mice are chimeras generatedby microinjection of fertilized eggs or by fusion of embryonic stemcells with megachromosome-containing cells, only cells from areas of themouse that carry the transgene are expected to express the transgene. Ifthe cells survive growth on hygromycin [or hygromycin and puromycin orneomycin, if the cells are obtained from mice generated by transfer ofboth antibiotic-resistance genes], this is one indication that they arestably expressing the transgenes. RNA isolated from the cells accordingto standard methods may also be analyzed by northern blot procedures todetermine if the cells express transcripts that hybridize to nucleicacid probes based on the antibiotic-resistance genes. Additionally,cells obtained from the transgenic mice may also be analyzed forβ-galactosidase expression using standard assays for this marker enzyme[for example, by direct staining of the product of a reaction involvingβ-galactosidase and the X-gal substrate, see, e.g., Jones (1986) EMBO5:3133-3142, or by measurement of β-galactosidase activity, see, e.g.,Miller (1972) in Experiments in Molecular Genetics pp. 352-355, ColdSpring Harbor Press]. Analysis of β-galactosidase expression isparticularly used to evaluate transgene expression in cells obtainedfrom control transgenic mice in which the only transgene transferredinto the embryo was the β-galactosidase gene.

Stable expression of the anti-HIV ribozyme gene in cells obtained fromthe transgenic mice may be evaluated in several ways. First, DNA--isolated from the cells according to standard procedures may besubjected to nucleic acid amplification using primers corresponding tothe ribozyme gene sequence. If the gene is contained within the cells,an amplified product of pre-determined size is detected uponhybridization of the reaction mixture to a nucleic acid probe based onthe ribozyme gene sequence. Furthermore, DNA isolated from the cells maybe analyzed using Southern blot methods for hybridization to such anucleic acid probe. Second, RNA isolated from the cells may be subjectedto northern blot hybridization to determine if the cells express RNAthat hybridizes to nucleic acid probes based on the ribozyme gene.Third, the cells may be analyzed for the presence of anti-HIV ribozymeactivity as described, for example, in Chang et al. (1990) Clin.Biotech. 2:23-31. In this analysis, RNA isolated from the cells is mixedwith radioactively labeled HIV gag target RNA which can be obtained byIn vitro transcription of gag gene template under reaction conditionsfavorable to in vitro cleavage of the gag target, such as thosedescribed in Chang et al. (1990) Clin. Biotech. 2:23-31. After thereaction has been stopped, the mixture is analyzed by gelelectrophoresis to determine if cleavage products smaller in size thanthe whole template are detected; presence of such cleavage fragments isindicative of the presence of stably expressed ribozyme.

b. Analysis of Whole Transgenic Mice

Whole transgenic mice that have been generated by transfer of theanti-HIV ribozyme gene [as well as selection and marker genes] intoembryos or fertilized eggs can additionally be analyzed for transgeneexpression by challenging the mice with infection with HIV. It ispossible for mice to be infected with HIV upon intraperitoneal injectionwith high-producing HIV-infected U937 cells [see, e.g., Locardi et al.(1992) J. Virol. 66:1649-1654]. Successful infection may be confirmed byanalysis of DNA isolated from cells, such as peripheral bloodmononuclear cells, obtained from transgenic mice that have been injectedwith HIV-infected human cells. The DNA of infected transgenic mice cellswill contain HIV-specific gag and env sequences, as demonstrated by, forexample, nucleic acid amplification using HIV-specific primers. If thecells also stably express the anti-HIV ribozyme, then analysis of RNAextracts of the cells should reveal the smaller gag fragments arising bycleavage of the gag transcript by the ribozyme.

Additionally, the transgenic mice carrying the anti-HIV ribozyme genecan be crossed with transgenic mice expressing human CD4 (i.e., thecellular receptor for HIV) [see Gillespie et al. (1993) Mol. Cell. Biol.13:2952-2958; Hanna et al. (1994) Mol. Cell. Biol. 14:1084-1094; andYeung et al. (1994) J. Exp. Med. 180:1911-1920, for a description oftransgenic mice expressing human CD4]. The offspring of these crossedtransgenic mice expressing both the CD4 and anti-HIV ribozyme transgenesshould be more resistant to infection [as a result of a reduction in thelevels of active HIV in the cells] than mice expressing CD4 alone[without expressing anti-HIV ribozyme].

4. Development of Transgenic Chickens Using Artificial Chromosomes

The development of transgenic chickens has many applications in theimprovement of domestic poultry, an agricultural species of commercialsignificance, such as disease resistance genes and genes encodingtherapeutic proteins. It appears that efforts in the area of chickentransgenesis have been hampered due to difficulty in achieving stableexpression of transgenes in chicken cells using conventional methods ofgene transfer via random introduction into recipient cells. Artificialchromosomes are, therefore, particularly useful in the development oftransgenic chickens because they provide for stable maintenance oftransgenes in host cells.

a. Preparation of Artificial Chromosomes for Introduction of Transgenesinto Recipient Chicken Cells

(i) Mammalian Artificial Chromosomes

Mammalian artificial chromosomes, such as the SATACs and minichromosomesdescribed herein, can be modified to incorporate detectable reportergenes and/or transgenes of interest for use in developing transgenicchickens. Alternatively, chicken-specific artifical chromosomes can beconstructed using the methods herein. In particular, chicken artificialchromosomes [CACs] can be prepared using the methods herein forpreparing MACs; or, as described above, the chicken librarires can beintroduced into MACs provided herein and the resulting MACs introducedinto chicken cells and those that are functional in chicken cellsselected.

As described in Examples 4 and 7, and elsewhere herein, artificialchromosome-containing mouse LMTK⁻-derived cell lines, orminichromosome-containing cell lines, as well as hybrids thereof, can betransfected with selected DNA to generate MACs [or CACs] that haveintegrated the foreign DNA for functional expression of heterologousgenes contained within the DNA.

To generate MACs or CACs containing transgenes to be expressed inchicken cells, the MAC-containing cell lines may be transfected with DNAthat includes λ DNA and transgenes of interest operably linked to apromoter that is capable of driving expression of genes in chickencells. Alternatively, the minichromosomes or MACs [or CACs], produced asdescribed above, can be isolated and introduced into cells, followed bytargeted integration of selected DNA. Vectors for targeted integrationare provided herein or can be constructed as described herein.

Promoters of interest include constitutive, inducible and tissue (orcell)-specific promoters known to those of skill in the art to promoteexpression of genes in chicken cells. For example, expression of thelacZ gene in chicken blastodermal cells and primary chicken fibroblastshas been demonstrated using a mouse heat-shock protein 68 (hsp 68)promoter [phspPTlacZpA; see Brazolot et al. (1991) Mol. Reprod. Devel.30:304-312], a Zn²⁺-inducible chicken metallothionein (cMt) promoter[pCBcMtlacZ; see Brazolot et al. (1991) Mol. Reprod. Devel. 30:304-312],the constitutive Rous sarcoma virus and chicken β-actin promoters intandem [pmiwZ; see Brazolot et al. (1991) Mol. Reprod. Devel.30:304-312] and the constitutive cytomegalovirus (CMV) promoter. Ofparticular interest herein are egg-specific promoters that are derivedfrom genes, such as ovalbumin and lysozyme, that are expressed in eggs.

The choice of promoter will depend on a variety of factors, including,for example, whether the transgene product is to be expressed throughoutthe transgenic chicken or restricted to certain locations, such as theegg. Cell-specific promoters functional in chickens include thesteroid-responsive promoter of the egg ovalbumin protein-encoding gene[see Gaub et al. (1987) EMBO J. 6:2313-2320; Tora et al. (1988) EMBO J.7:3771-3778; Park et al. (1995) Biochem. Mol. Biol. Int. (Australia)36:811-816].

(ii) Chicken Artificial Chromosomes

Additionally, chicken artificial chromosomes may be generated usingmethods described herein. For example, chicken cells, such as primarychicken fibroblasts [see Brazolot et al. (1991) Mol. Reprod. Devel.30:304-312], may be transfected with DNA that encodes a selectablemarker [such as a protein that confers resistance to antibiotics] andthat includes DNA (such as chicken satellite DNA) that targets theintroduced DNA to the pericentric region of the endogenous chickenchromosomes. Transfectants that survive growth on selection medium arethen analyzed, using methods described herein, for the presence ofartificial chromosomes, including minichromosomes, and particularlySATACs. An artificial chromosome-containing transfectant cell line maythen be transfected with DNA encoding the transgene of interest [fusedto an appropriate promoter] along with DNA that targets the foreign DNAto the chicken artificial chromosome.

b. Introduction of Artificial Chromosomes Carrying Transgenes ofInterest into Recipient Chicken Cells

Cell lines containing artificial chromosomes that harbor transgene(s) ofinterest (i.e., donor cells) may be fused with recipient chicken cellsin order to transfer the chromosomes into the recipient cells.Alternatively, the artificial chromosomes may be isolated from the donorcells, for example, using methods described herein [see, e.g., Example10], and directly introduced into recipient cells.

Exemplary chicken recipient cell lines include, but are not limited to,stage X blastoderm cells [see, e.g., Brazolot et al. (1991) Mol. Reprod.Dev. 30:304-312; Etches et al. (1993) Poultry Sci. 72:882-889; Petitteet al. (1990) Development 108:185-189] and chick zygotes [see, e.g.,Love et al. (1994) Biotechnology 12:60-63].

For example, microcell fusion is one method for introduction ofartificial chromosomes into avian cells [see, e.g., Dieken et al.[(1996) Nature Genet. 12:174-182 for methods of fusing microcells withDT40 chicken pre-B cells]. In this method, microcells are prepared [forexample, using procedures described in Example 1.A.5] from theartificial chromosome-containing cell lines and fused with chickenrecipient cells.

Isolated artificial chromosomes may be directly introduced into chickenrecipient cell lines through, for example, lipid-mediated carriersystems, such as lipofection procedures [see, e.g., Brazolot et al.(1991) Mol. Reprod. Dev. 30:304-312] or direct microinjection.Microinjection is generally preferred for introduction of the artificialchromosomes into chicken zygotes [see, e.g., Love et al. (1994)Biotechnology 12:60-63].

c. Development of Transgenic Chickens

Transgenic chickens may be developed by injecting recipient Stage Xblastoderm cells (which have received the artificial chromosomes) intoembryos at a similar stage of development [see, e.g., Etches et al.(1993) Poultry Sci. 72:882-889; Petitte et al. (1990) Development108:185-189; and Carsience et al. (1993) Development 117: 669-675]. Therecipient chicken embryos within the shell are candled and allowed tohatch to yield a germline chimeric chicken that will express thetransgene(s) in some of its cells.

Alternatively, the artificial chromosomes may be introduced into chickzygotes, for example through direct microinjection [see, e.g., Love etal. (1994) Biotechnology 12:60-63], which thereby are incorporated intoat least a portion of the cells in the chicken. Inclusion of atissue-specific promoter, such an an egg-specific promoter, will ensureappropriate expression of operatively-linked heterologous DNA.

The DNA of interest may also be introduced into a minichromosome, bymethods provided herein. The minichromosome may either be one providedherein, or one generated in chicken cells using the methods herein. Theheterologous DNA will be introduced using a targeting vector, such asthose provided herein, or constructed as provided herein.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method for producing a cell that contains heterologous nucleicacid, comprising: introducing a satellite artificial chromosome (SATAC)into the cell.
 2. The method of claim 1, wherein the cell is an animalcell.
 3. The method of claim 2, wherein the cell is a mammalian cell. 4.The method of claim 1, wherein the SATAC is a mammalian artificialchromosome.
 5. The method of claim 1, wherein the SATAC is a plantartificial chromosome.
 6. The method of claim 1, wherein the SATAC isintroduced by a method selected from direct DNA transfer or uptake,electroporation, lipofection, nuclear microinjection, liposome mediatedtransfer, microprojectile bombardment, polycation-mediated transfer andmicrocell fusion.
 7. The method of claim 1, wherein the SATAC isintroduced by a method selected from microinjection, nuclear transfer,electrofusion, projectile bombardment and lipid-mediated transfersystems.
 8. The method of claim 1, wherein the SATAC is introduced intothe cell by a method, comprising: introducing a DNA fragment into thecell, wherein the DNA fragment comprises a selectable marker; growingthe cell under selective conditions to produce cells that haveincorporated the DNA fragment into their genomic DNA; and selecting acell that comprises a satellite artificial chromosome (SATAC).
 9. Themethod of claim 8, wherein the DNA fragment is introduced into oradjacent to an amplifiable region of a chromosome in the cell.
 10. Themethod of claim 9, wherein the amplifiable region comprises rDNA. 11.The method of claim 9, wherein the amplifiable region comprisesheterochromatin.
 12. The method of claim 8, wherein the DNA isintroduced into pericentric heterochromatin in a chromosome of the cell.13. The method of claim 8, wherein the method for producing the SATACfurther comprises isolating the SATAC.
 14. The method of claim 8,wherein in the method for producing the SATAC, the DNA fragmentcomprises a sequence of nucleotides that targets the fragment to theheterochromatic region of a chromosome.
 15. The method of claim 14,wherein the targeting sequence of nucleotides comprises satellite DNA.16. The method of claim 9, wherein, in the method for producing theSATAC, the cell is a mammalian cell, a plant cell, a fish cell, aninsect cell, a reptilian cell, an amphibian cell, an arachnid cell or arodent cell.
 17. The method of claim 1, wherein the SATAC is an isolatedsubstantially pure SATAC.
 18. The method of claim 1, wherein the SATACis a megachromosome, comprising about 50 to about 450 megabases (Mb).19. The method of claim 1, wherein the SATAC is a megachromosome,comprising about 250 to about 400 Mb.
 20. The method of claim 1, whereinthe SATAC is a megachromosome, comprising about 150 to about 200 Mb. 21.The method of claim 1, wherein the SATAC is a megachromosome, comprisingabout 90 to about 120 Mb.
 22. The method of claim 1, wherein the SATACis a megachromosome, comprising about 15 to about 60 Mb.
 23. The methodof claim 1, wherein the cell is an embryonic cell.
 24. The method ofclaim 1, wherein the cell is an embryonic stem cell.
 25. The method ofclaim 1, wherein the SATAC comprises nucleic acid encoding a geneproduct or a plurality of gene products.
 26. The method of claim 25,wherein the gene products comprise a biosynthetic pathway.
 27. Themethod of claim 2, wherein the animal is a mammal, bird, fowl, fish,invertebrate, vertebrate, reptile or insect.
 28. The method of claim 1,wherein the cell is a mammalian cell, a plant cell, a fish cell, aninsect cell, a reptilian cell, an amphibian cell, an arachnid cell or arodent cell.